Extending molecular imaging into the shortwave-infrared (SWIR, 900-1400 nm) region provides deep tissue visualization of biomolecules in the living system resulting from the low tissue autofluorescence and scattering. Looking at the Food and Drug Administration-approved and clinical trial near-infrared (NIR) probes, only indocyanine green (ICG) and its analogues have been approved for biomedical applications. Excitation wavelength less than 800 nm limits these probes from deep tissue penetration and noninvasive fluorescence imaging. Herein, we present the synthesis of ICG-based π-conjugation-extended cyanine dyes, ICG-C9 and ICG-C11 as biocompatible, and water-soluble SWIR-emitting probes with emission wavelengths of 922 and 1010 nm in water, respectively. Also, ICG-, ICG-C9-, and ICG-C11-based fluorescent labeling agents have been synthesized for the development of SWIR molecular imaging probes. Using the fluorescence of ICG, ICG-C9, and ICG-C11, we demonstrate three-color SWIR fluorescence imaging of breast tumors by visualizing surface receptors (EGFR and HER2) and tumor vasculature in living mice. Furthermore, we demonstrate two-color SWIR fluorescence imaging of breast tumor apoptosis using an ICG-conjugated anticancer drug, Kadcyla and ICG-C9 or ICG-C11-conjugated annexin V. Finally, we show long-term (38 days) SWIR fluorescence imaging of breast tumor shrinkage induced by Kadcyla. This study provides a general strategy for multiplexed fluorescence molecular imaging with biocompatible and water-soluble SWIR-emitting cyanine probes.
Keywords: breast cancer; fluorescence imaging; molecular probe; multiplexed imaging; organic dye; shortwave-infrared.