NIR-II Fluorescent Timer-Embedded Drug for Real-Time Tracing of Immunogenic Cell Death and Guiding Chemo-immunotherapy

Immune adjuvants are essential components in chemo-immunotherapy; however, determining the optimal administration timing to synchronize them with recruited immune cells is challenging due to the difficulty in <i>in vivo</i> tracing of immunogenic cell death (ICD). Current <i>ex vivo</i> ICD assessment methodologies delay reporting and are thus incapable of guiding <i>in situ</i> chemo-immunotherapy. Here, we develop a cell Membrane-Anchored NIR-II Fluorescent Timer-Embedded Drug (MANTED) to real-time track ICD and guide adjuvant administration timing. MANTED is constructed by conjugating a DNA NIR-II ATP reporter to rare earth nanoparticles (RENPs), loading with chemotherapy drugs to the DNA helix, and anchoring to a 4T1 cell membrane via a biorthogonal click reaction, which prevents endocytosis and intracellular ATP interference. The ATP reporter is functionalized with the quencher FD1080 to quench NIR-II fluorescence of RENPs and is blocked with a photocleavable complementary strand to suppress tumor microenvironment ATP interference before the ICD process. Upon 980 nm laser irradiation, the UV upconversion emission of RENPs cleaves the PC linker, releasing the drug for chemotherapy and activating MANTED. Subsequent 808 nm laser irradiation induces RENPs' downconversion NIR-II fluorescence recovery upon ATP recognition, which is monitored in real time. The optimal timing for adjuvant administration is chosen when the fluorescence intensity is saturated. MANTED-guided chemo-immunotherapy enhances therapeutic efficiency.