Controlled In Situ Self‐Assembly for Pretargeted Tumor Theranostics via Bioorthogonal Activation

Activatable phototheranostic agents that integrate fluorescence imaging with photodynamic therapy (PDT) represent a promising strategy for cancer treatment. However, conventional agents often suffer from nonspecific activation and uncontrolled pharmacokinetics, which limit their ability to achieve precise theranostics. To address this, we present an ultra-specific in situ activatable platform for tumor phototheranostics that integrates controlled pretargeting via in situ self-assembly with bioorthogonal reaction-mediated activation. This platform consists of two key components: a cathepsin B and glutathione dual-responsive pretargeting agent, Val-Cit-Cys(StBu)-Lys(Tz)-CBT (CBT-Tz), which undergoes tumor microenvironment-triggered self-assembly into nanoaggregates, leading to localized accumulation of tetrazine (Tz) groups; and a vinyl ether-caged and PEGylated hemicyanine (PEG-hCy-VE) as an activatable theranostic agent, which can be uncaged via a bioorthogonal activation with the pre-accumulated Tz groups. This reaction restores near-infrared fluorescence and activates potent PDT activity selectively at the tumor site. The pretargeting strategy, coupled with the activation approach, provides superior spatiotemporal control, enabling simultaneous high-contrast fluorescence imaging and spatially precise PDT. Guided by real-time imaging, personalized PDT was implemented, leading to effective tumor suppression with minimal systemic toxicity. By synergizing in situ self-assembly and bioorthogonal activation, this work provides a robust, extensible, and highly specific platform for precise drug delivery and heterogeneous tumor theranostics.