Rapid and Specific Cell-Surface Anchoring of Gold Nanostars for Photoacoustic BMSC Imaging

Background: Bone marrow mesenchymal stem cell (BMSC) therapy holds great promise for regenerative medicine, but its clinical translation is hindered by the lack of non-invasive, real-time methods to track transplanted cell fate in vivo. Although photoacoustic (PA) imaging offers deep-tissue penetration and high sensitivity, existing cell-labeling strategies relying on endocytosis of contrast agents have drawbacks of prolonged incubation times and variable efficiency, thus potentially compromising cell viability and function. Methods: To address these limitations, we developed a rapid, bioorthogonal pre-labeling strategy. Azide (N_3) groups were first metabolically engineered onto BMSC surfaces. Subsequently, dibenzocyclooctyne (DBCO)-functionalized gold nanostars (Au-star-DBCO) were conjugated to the cells via a highly efficient, copper-free click reaction, thereby enabling covalent membrane anchoring. Results: This approach achieved rapid and specific stem cell labeling within 3 h, with an efficiency of 83.1% ± 0.67. The labeling process did not impair BMSC viability or multilineage differentiation potential. The Au-star-DBCO-labeled BMSCs generated strong, concentration-dependent PA signals both in vitro and in a rat subcutaneous model, and enabled dynamic monitoring for at least 72 h post-transplantation. The specificity of the bioorthogonal reaction provided a significantly higher signal-to-noise ratio than passive uptake methods. Conclusions: We successfully established a biocompatible and efficient platform for stem cell tracking by integrating metabolic glycoengineering, bioorthogonal chemistry, and nanostar-enhanced PA imaging. This strategy overcomes key limitations of traditional endocytosis-based labeling, and offers a rapid, specific, and functional cell-compatible tool for sensitive, longitudinal in vivo monitoring. This platform may advance precise assessment of cell therapies and facilitate their clinical application.