Semi-Quantitative In Vivo Imaging of Intratumorally Injected Iodine-125-Labeled Nanoparticles Using a Benchtop 2D Multi-Pinhole X-ray System

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 ( 125 I)-labeled gold (Au) core and silver (Ag) shell NPs (PEG- 125 I-Au@Ag NPs) were used. The multi-pinhole X-ray imaging system was calibrated using PEG- 125 I-Au@Ag NP-containing solutions, and the activity threshold was estimated. Three CT26 tumor-bearing BALB/c mice received intratumoral injections of 500 μCi PEG- 125 I-Au@Ag 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 2 = 0.9992, and the extrapolated activity threshold was 0.05 μCi. In vivo imaging tracked intratumoral retention kinetics, showing a marked decline within 24 h followed by a slower phase through 120 h. Endpoint ex vivo imaging supported sustained intratumoral retention at T = 120 h, with 26.41 ± 2.09% of the injected dose and 202.35 ± 98.83% of the injected dose per gram retained in the tumor. The liver activity was 1.17 ± 0.80% of the injected dose and 0.82 ± 0.64% of the injected dose per gram, whereas the thyroid activity was 1.92 ± 0.15% of the injected dose and 97.94 ± 20.11% of the injected dose per gram. The difference between in vivo and ex vivo measurements ranged from 0.03 to 3.51 μCi, with relative percent differences of 0.74% to 12.80%. Conclusions The multi-pinhole X-ray imaging system enables semi-quantitative assessment of intratumoral retention and biodistribution of PEG- 125 I-Au@Ag NPs in the measured organs, providing a practical approach for longitudinal preclinical evaluation.