Affibody-based optical imaging probe for noninvasive detection of liver fibrosis

Background: Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease affecting 2-6% of the global population, which can eventually lead to liver failure or cancer. The current standard for diagnosis is liver biopsy, which is invasive, subjective, painful, and carries potential complications. We present a novel non-invasive optical imaging approach using a miniprotein based probes (affibody) targeting collagen type-1 (COL-1), a histologically established marker of MASH, to detect liver fibrosis in animal models of MASH in vivo with high sensitivity and specificity. thereby enabling the potential of early diagnosis and longitudinal assessment of disease progression and response to treatments. Methods: We synthesized multiple affibody molecules with varying binding affinities to COL-1, two of which (called Clone 3 and 7) are reported here, and a non-binding control structure. We conjugated the affibodies with a near-infrared (NIR) fluorophore dye (Dy800), enabling the detection and monitoring of the probes in vivo using fluorescent optical imaging. We used two different MASH animal models, high-fat choline-deficient (HFCDA) diet-fed mice and Gubra-Amylin (GAN) mice, along with their age-matched normal chow-fed (NC-fed) controls, to assess the biodistribution, binding specificity, and liver accumulation of the COL-1 affibodies through in vivo whole-body optical imaging and ex vivo tissue imaging. Results: The affibody optical imaging probe had 50-fold higher affinity (~34 nM) compared to the state-of-the-art imaging probes (typically, > 1 µM). It demonstrated rapid blood clearance via renal elimination, with complete elimination observed at 48 h post-injection. Imaging data obtained from both animal models showed significantly higher liver fluorescence signal intensity compared to their age-matched NC-fed controls. Specificity was confirmed by comparison of images collected with binding and non-binding probes, the latter showing significantly lower accumulation in the liver. A strong correlation between histology-derived collagen content and ex vivo liver imaging data was found in diseased animals (r² = 0.86). Conclusions: In vivo imaging of liver fibrosis using collagen-targeting affibody probes offers a promising non-invasive alternative to liver biopsy, potentially improving diagnosis accuracy and accelerating drug development.