Homologous Membrane‐Camouflaged Pretargeting Synergized with Bioorthogonal Amplification Enables High‐Fidelity Tumor Imaging

Abstract Bioorthogonal chemistry‐facilitated pretargeting offers significant potential for precise cancer diagnosis. Addressing the critical challenges of nonspecific labeling inherent in pretargeting reagents and substantially enhancing tumor anchoring efficiency is paramount. Herein, a biomimetic “Trojan horse” pretargeting strategy synergistically integrated with bioorthogonal cascade labeling is introduced for high‐fidelity tumor imaging. This approach achieves highly specific tumor targeting through 4T1‐biomimetic, ROS‐responsive nanocarriers (Az‐ROS@MB) delivering azide precursors (Ac 4 ManNAz), enabling spatiotemporally controlled metabolic incorporation of azide tags specifically at target sites, and ensuring powerful signal amplification via bioorthogonal cascade imaging with a glutathione (GSH)‐responsive nanoprobe (CSSC@DBCO). In vitro and in vivo validation demonstrated efficient azide labeling by Az‐ROS@MB, specific bioorthogonal targeting with CSSC@DBCO, and significant imaging probe enrichment in orthotopic 4T1 tumors, highlighting the strategy's precision and efficacy. The synergy of “biomimetic‐responsive‐click” cascade pretargeting and bioorthogonal signal amplification enables precise tumor cell labeling, enhancing pretargeted reporter enrichment for prolonged tumor retention and high‐contrast imaging.