Self‐Included Fluorescent Probes via Azocalix[4]arene–Dye Conjugation for Activatable and Stable NIR Imaging

Near-infrared (NIR) fluorescent probes are crucial tools in bioimaging; however, the challenge of improving their stability and targeting precision remains a key issue. In this work, we present a novel strategy for designing Self-Included Fluorescent Probes (SIFPs) using a covalent macrocyclic host-dye conjugate approach. By covalently linking NIR dyes, such as indole heptamethine cyanine fluorescent probes, to azocalix[4]arene carriers, we leverage the unique properties of the macrocyclic host to encapsulate and protect the dyes intramolecularly, while enhancing their stability and preventing off-target leakage. Importantly, the azocalixarene's hypoxia-responsive azo linkers enable targeted activation of the dye in tumor environments, improving imaging accuracy. Through comprehensive in vitro and in vivo experiments, we demonstrate that SIFPs significantly outperform conventional probes and their host-guest binary complexes in terms of stability and imaging precision. This strategy not only combines the benefits of covalent and non-covalent binding but also offers an effective method for enhancing the design of activatable, stable fluorescent probes.