Hypoxia Imaging in Hepatic Fibrosis: A Preliminary Application of CAIX‐Targeted MRI Probe in Rats

ABSTRACT This study aimed to validate the feasibility of hypoxia‐targeted magnetic resonance imaging (MRI) molecular probe (CAIX‐Gd) for evaluating hypoxia status in rats with hepatic fibrosis. A macrocyclic ligand compound (Compound 3) was synthesized using the carbonic anhydrase IX (CAIX) inhibitor acetazolamide as the targeting moiety, and its structure was confirmed by 1 H‐NMR spectroscopy. CAIX‐Gd was then prepared by chelating gadolinium, with free gadolinium content quantified via the xylenol orange assay. In biocompatibility tests, CAIX‐Gd administration showed no significant histopathological changes compared with saline controls. For hypoxia detection, rats were divided into three groups: (A) hepatic fibrosis (HF) with CAIX‐Gd, (B) HF with DTPA‐Gd, and (C) normal controls (NC) with CAIX‐Gd. The difference in T1 relaxation time (∆T1) pre‐ and post‐contrast was measured in regions of interest (ROI). HF rats showed increased collagen deposition (Masson staining), α‐SMA activation, and elevated hypoxia markers (PIMO/HIF‐1α) compared to controls (all p < 0.05). In group C, mild ∆T1 shortening was seen at 10 min post‐injection. While ∆T1 continuously decreased in group B, the shortening was significantly greater in group A ( p < 0.05), peaking at 10 min. In conclusion, the CAIX‐Gd probe was successfully synthesized and demonstrated potential for the non‐invasive MRI detection of hypoxia in hepatic fibrosis.