Construction of Matrix Metalloproteinase-13-Activatable cRGD-Targeted Ratiometric Fluorescent Nanoprobe for Imaging Microscopic Osteosarcoma Lesions and Pulmonary Metastases

Abstract As an important matrix metalloproteinase (MMP) family member, MMP-13 is involved in collagen degradation, extracellular matrix remodeling, and tumor invasion and metastasis, which represents a promising molecular target for activity-based detection of osteosarcoma (OS)-associated lesions. Based on this feature, an MMP-13-activatable, cyclic Arg-Gly-Asp (cRGD)-targeted ratiometric fluorescent nanoprobe, UCNP@PDA@Cy5-pep-cRGD, has been constructed through immobilization of Cy5 modified MMP-13 peptide substrate with cRGD motif (Cy5-pep-cRGD) on the polydopamine coated upconversion nanoparticle (UCNP@PDA). In this nanoprobe, UCNP is employed as a stable upconversion luminescence (UCL) reference, polydopamine (PDA) is used as a low-background fluorescence-quenching platform, Cy5 is modified on an MMP-13-cleavable peptide substrate as the responsive signal, and cRGD is further modified on the nanoprobe surface to enhance its tumor-targeted accumulation. The nanoprobe has been systematically characterized, and evaluated for MMP-13 responsiveness, selectivity, ratiometric detection performance, and biological stability. Cellular experiments demonstrate that UCNP@PDA@Cy5-pep-cRGD has strong targeted accumulation and enzyme-responsive imaging capability toward tumor cells. In vivo and ex vivo imaging experiments further confirm its ability to visualize MMP-13-responsive activation in microscopic OS lesions and pulmonary metastases. This strategy enables in situ visualization of MMP-13-mediated nanoprobe activation for the identification of microscopic OS lesions and pulmonary metastases.