Protein-encapsulation-driven high-performance luminogen for three-photon imaging of deep tumor in the NIR-II biowindow

The development of organic molecules activated by near-infrared II (NIR-II) light holds considerable potential for deep image-guided disease diagnosis and therapeutic applications. However, the design and synthesis of such materials remain a significant challenge due to limitations in controllable molecular architecture. In this study, two small molecules, TPEPyC2 and DTPEPyC5 , were firstly synthesized. Subsequently, their respective assemblies, namely TPEPyC2@HSA and DTPEPyC5@HSA , were obtained through co-assembled with the hydrophobic human serum albumin (HSA). Comprehensive analyses demonstrated that only DTPEPyC5@HSA exhibited superior photostability, biosafety, and a large three-photon absorption cross-section (3.85 × 10 −80  cm 6  s 2 ). Notably, this probe enabled precise mitochondrial targeting in live HepG2 cells, as indicated by a co-localization coefficient of Pr  = 0.88, and achieved tumor tissue penetration of 143 μm using three-photon fluorescence imaging. This bio-protein encapsulation strategy provides a novel molecular design framework for developing NIR–II–excitable fluorescent probes, thereby significantly enhancing their imaging performance.