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

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.