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

ABSTRACT 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.