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Purpose: The purpose of this study was to show a systematic strategy for assessing the pharmacokinetics of indocyanine green (ICG)-loaded nanoparticles in the tumor tissue based on a dynamic diffuse fluorescence tomography (DFT) system.Procedures: Twelve-seven-week-old male Balb/c nude mice bearing HepG2/ADR hepatocellular carcinoma were randomly divided into four groups (n = 3 per group). Four hundred microliters of three types of ICG-loaded nanoparticles (content of ICG: 50 μg/ml) and free ICG (50 μg/ml) was intravenously injected into the mice in each group, respectively. Afterwards, the real-time tomographic images on the spatial level were acquired at 2-11 min, 30 min, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h post-injection, and pharmacokinetic rates were derived for semi-quantitative assessment of the pharmacokinetics of nanoparticles at the tumor site using our proposed pharmacokinetic analysis method.Results: The results obtained from our proposed dynamic DFT experiment demonstrated the distribution of different ICG formulations on the spatial level and enabled the semi-quantitative analysis of the pharmacokinetics of nanoparticles in the tumor tissue.Conclusions: The obtained pharmacokinetic rates effectively reflected the metabolic processes of nanoparticles in the tumor tissue, which proves to be beneficial for the development of tumor diagnosis and therapy. [ABSTRACT FROM AUTHOR]