Image-guided in vivo evaluation and comparison of bone-targeting peptides for therapeutic intervention.

Hydroxyapatite (HA)-binding peptides hold strong potential for bone-targeted therapies due to their high affinity for mineralized tissues. However, most existing studies have primarily focused on in vitro binding characterization, offering limited insight into their in vivo biodistribution and bone-binding performance. In this study, we evaluated the in vivo behavior of four HA-binding peptides, including D8, E8, YD8, and YE8, using fluorescence imaging to assess biodistribution in both healthy and pathological bone environments. In healthy animals, D8 showed the strongest bone-binding capacity, with prominent localization in the skull, femur, and tibia, while YD8 exhibited moderate binding. E8 and YE8 showed more limited localization, influenced by peptide dosage and binding kinetics. In pathological models, including tibial defects and osteogenesis imperfecta (OIM) mice, D8 and YD8 preferentially accumulated in compromised bone regions, highlighting their potential utility in targeting diseased bone microenvironments. Fluorescence imaging combined with spectral unmixing algorithms enabled effective visualization and quantification of peptide localization and distribution. These findings emphasize the value of in vivo studies for advancing the therapeutic and diagnostic applications of HA-binding peptides. The results provide a foundation for optimizing peptide design to improve specificity and efficacy in bone repair and regeneration.