Globally, hepatobiliary cancers including hepatocellular carcinoma (HCC) and intra/extrahepatic cholangiocarcinoma are highly lethal cancers, with pathological conditions such as liver cirrhosis and chronic hepatitis being the most important risk factors. Most patients with HCC are already in an advanced stage at the time of diagnosis, and there is therefore an unmet need for accurate early diagnosis of HCC nodules, whereby curative treatments are still available. Hepatobiliary magnetic resonance imaging (MRI) has been advocated by clinical guidelines as the most sensitive method for non-invasive diagnosis of liver tumours in patients, whereby hepatocyte-specific contrast agents (CAs) are used to selectively modulate MR signals to improve tumour visibility. Since early HCCs with a typical diameter smaller than 1.5 cm present as vague and poorly vascularized nodules with indistinct margins, and are characterized at the molecular and cellular levels by progressive accumulation of epigenetic and genetic alterations, successful in vivo MR detection of early HCCs rely particularly on sensitive and specific CAs for reliable visualization of subtle molecular alternations in the complex liver system.

 Currently, gadoxetate disodium (Gd-EOB-DTPA) is the primary choice for usage in hepatobiliary MRI of HCCs in the clinics. In such a system, efficient visualization of the liver lesions is facilitated by specific binding of the ethoxybenzyl (EOB) ligands with organic anion transporter polypeptides (OATPs) present on the membranes of the hepatocytes, which display varied expression levels between HCCs and the surrounding healthy tissue. However, due to the low relaxivity and poor specificity, the performance of hepatobiliary MRI using Gd-EOB-DTPA in hepatic tumours is limited. For example, it is difficult for detection of HCC with ~1 cm in diameter. Thus, approximately 53% of tumours with 1 to 2 cm in size are not accurately diagnosed, where intrahepatic spread can occur. Moreover, about 5%-12% of moderately differentiated HCCs may overexpress OATPs aberrantly, resulting in loss of efficacy of Gd-EOB-DTPA. In addition, the gadolinium-based CAs are also known to induce nephrogenic fibrosis in renal dysfunction patients, and potential long-term accumulation in human bone and brain, raising considerable safety concerns.

 Nanoparticles are now emerging as promising candidates for high performance molecular imaging due to their small size and functional versatility. However, the design of hepatocyte-specific nanoparticles has long been a challenge because most nanoparticles are likely to be sequestered by Kupffer cells regardless of their surface ligands. Here, we report an ultrasmall manganese ferrite nanoparticle modified with polyethylene glycol-ethoxy-benzyl ligand on the surface (MnFe₂O₄-EOB-PEG), with superior hepatocyte specificity for sensitive and high-resolution hepatobiliary MRI as demonstrated in large animals, including pigs, rabbits, and the more clinically relevant macaques. We observed that synergistic collaboration of the 3 nm MnFe₂O₄ core and PEG-EOB ligand in the MnFe₂O₄-EOB-PEG, enabled precise hepatocyte specificity co-mediated by OATP1B1/B3 and the identified SLC39A14. Such a high hepatocyte specificity of MnFe₂O₄-EOB-PEG consequently lead to rapid (10 min versus 30 min) and high-resolution hepatobiliary MRI (where hepatic ducts with diameter as small as 0.5 mm can be identified), with a significantly higher detection sensitivity (92% versus 48%) of sub-5 mm liver tumours in rabbits, and an even more accurate assessment of biliary obstruction in macaques, as compared to the clinically used hepatobiliary agent, Gd-EOB-DTPA. Moreover, results from preclinical safety evaluation in pigs showed that MnFe₂O₄-EOB-PEG is highly biocompatible, The MnFe₂O₄-EOB-PEG was excreted quickly in the form of intact nanoparticles via hepatic clearance (88.26%) and renal clearance (11.59%) pathways, with only < 1% of administered MnFe₂O₄-EOB-PEG that continued to linger after 7 days.

 Together, we have provided ample evidence that biocompatible MnFe₂O₄-EOB-PEG, has significant advantages over clinically used Gd-EOB-DTPA for non-invasive visualization of small liver tumours and hepatobiliary tract related diseases, thus, showing great potential in clinical translation as high-performance liver-specific MRI contrast agents.