Off–On–Off Probes for Precise Theranostics

Smart probes are crucial for precise theranostics. The current focus of most research in precise theranostics has mainly centered on the activation process of smart probes after intravenous injection into target tissues. However, subsequent events following the probe activation within target tissues are typically overlooked. Of particular note is the underexplored aspect of activated probe diffusion from diseased to normal tissues. Herein, we present the latest development on off-on-off probes, highlighting not only their activation upon intravenous injection into target tissues but also the subsequent diffusion of activated probes from target to normal tissues. In particular, we focus on molecular design strategies to achieve off-on-off signal control, including approaches such as intramolecular donor-acceptor reconfiguration, assembly, disassembly, Förster resonance energy transfer, molecular structure tautomeric forms, dynamic steric effects, biodegradation, and multimechanism combination. We provide a comprehensive summary of how these probes have been evaluated in preclinical models, focusing on their advantages over conventional off-on probes in enhancing imaging specificity and reducing off-target therapeutic side effects. Finally, we offer forward-looking insights into the advancement of off-on-off theranostic probes, their broader integration into biomedical applications, and the challenges that must be addressed for practical implementation.