Targeted Near‐Infrared Photoacoustic Probes for Dual‐Channel Cartilage and Bone Imaging

ABSTRACT Simultaneous imaging of cartilage and bone with molecular specificity remains a significant unmet need in orthopedic research and intraoperative guidance. Here, we report the development of two tissue‐targeted near‐infrared photoacoustic probes (Cart‐670 for cartilage and Osteo‐750 for bone) that enable dual‐channel visualization of these adjacent tissues within a single imaging session. The probes were designed through a modular strategy: A systematic screen of NIR‐absorbing non‐fluorescent dyes identified QSY21 as the optimal photoacoustic scaffold (limit of detection: 0.5 µM in PBS; signal‐to‐noise ratio ≥ 3), which was then functionalized with a cationic cartilage‐targeting motif to yield Cart‐670. In parallel, Osteo‐750 was synthesized by conjugating a bisphosphonate moiety to Alexa Fluor 750 for bone targeting. Both probes exhibit strong NIR absorption, low detection limits (Cart‐670: 1 µM; Osteo‐750: 2–3 µM in PBS), and selective ex vivo tissue accumulation: Cart‐670 shows preferential retention in cartilage over bone, while Osteo‐750 produces ∼200× higher photoacoustic signal on bone relative to cartilage. Spectral unmixing at 680 and 750 nm enables artifact‐free two‐color imaging without cross talk, demonstrating the feasibility of multiplexed photoacoustic visualization of the cartilage‐bone interface.