A Tandem‐Responsive and Multifunctional Dendritic Dot for Acid and γ‐Glutamyl Transpeptidase‐Activated Cancer Theranostics

Stimuli-responsive "off-on" theranostic nanoprobes enable precise cancer diagnosis and treatment by activating functions within the tumor microenvironment. Current challenges include selectivity, multi-functionality, and structural definition. Here, we report a tandem-responsive multiple-functional (TRIPLE) dendritic dot (DD) synthesized via a single-molecule all-in-one (SMALL) strategy. This TRIPLE DD integrates tumor-responsive drug release, fluorescence activation for imaging, and transcytosis for deep tumor penetration. Utilizing a polylysine dendrimer with a Cy5 core and covalently conjugated camptothecin (CPT), Cy5 fluorescence is initially quenched by CPT via photoinduced electron transfer (PET) ("off" state). A surface β-carboxylic amide-linked γ-glutamylglycine motif enables sequential cleavage by acidic pH and γ-glutamyl transpeptidase (GGT), exposing amines. These amines catalyze CPT release and activate Cy5 fluorescence. The resulting cationic DD induces cellular transcytosis, enhancing drug infiltration. We demonstrate pH/GGT tandem-responsive drug release, fluorescence activation, and potent antitumor activity in vitro and in vivo, notably against hepatocellular carcinoma. This work represents a rational design of multifunctional stimuli-responsive off-on nanoprobes, paving the way for advanced precision theranostics.