Collagen IV-targeted phase-change nanoparticles illuminate early liver fibrosis staging <i>via</i> ultrasound molecular imaging

Early non-invasive diagnosis of liver fibrosis remains a significant clinical challenge. This study aimed to develop type IV collagen-targeted phase-change nanoparticles (AC-IV-PFP@NPs) for ultrasound molecular imaging (UMI), allowing accurate staging of early-stage liver fibrosis. AC-IV-PFP@NPs were prepared by conjugating anti-collagen IV antibody (AC-IV) to perfluoropentane-encapsulated liposomes <i>via</i> carbodiimide coupling. Physicochemical properties were characterized using transmission electron microscopy, dynamic light scattering, and confocal microscopy. In CCl₄-induced fibrotic rats representing METAVIR stages S0-S4, the targeted nanoparticles were administered intravenously. The nanoparticles displayed spherical morphology with a mean diameter of 307.92 ± 4.16 nm, high AC-IV conjugation efficiency (78.94 ± 2.83%), and a favorable biosafety profile (cell viability >87% at 6 mg mL^-1). Targeting specificity was validated both <i>in vitro</i> and <i>in vivo</i>, with fluorescence imaging showing a 3.8-fold increase in binding to fibrotic collagen IV relative to non-targeted controls (<i>P</i> < 0.001). CEUS signal intensity peaked at 30 min post-injection and showed a strong positive correlation with the fibrosis stage (<i>r</i> = 0.725, <i>P</i> < 0.001). ROC analysis demonstrated high diagnostic accuracy for early fibrosis: an area under the curve (AUC) of 0.949 for distinguishing S0 from S1-S4 (sensitivity 85.5%, specificity 91.7%) and an AUC of 0.923 for separating S0-S1 from S2-S4 (sensitivity 90.7%, specificity 79.2%). To date, AC-IV-PFP@NPs represent the first type IV collagen-targeted UMI platform for liver fibrosis staging in rats, offering non-invasive, real-time assessment with high sensitivity for early-stage disease (S1-S2). This approach addresses the limitations of biopsy and conventional imaging and offers a promising and transformative approach for clinical fibrosis management.