Quantitative Tracking of Apoptotic Caspase‐3 In Vivo for Early Evaluation of Radiation Therapy Efficacy

Abstract Traditional responsive fluorescent probes are predominantly restricted to qualitative biomarker detection, incapable of delivering real‐time quantitative analysis or spatial mapping of protease activity in vivo, which is essential for elucidating disease progression. To overcome this, a ratiometric second near‐infrared region (NIR‐II) fluorescent (FL) probe (DCNP@IR‐806) was developed by conjugating caspase‐3‐specific peptide substrates and sensitizer molecules (IR‐806) to lanthanide‐doped down‐conversion nanoparticles (DCNP). DCNP@IR‐806 achieves single‐channel emission at 1550 nm under dual excitation, facilitating self‐calibrated quantification and real‐time monitoring of activated caspase‐3 in vivo. Radiotherapy induces tumor cell apoptosis, thereby activating caspase‐3, which subsequently triggers a ratiometric NIR‐II FL signal change of DCNP@IR‐806. The ratiometric signal demonstrates a linear correlation with caspase‐3 concentration, achieving a detection limit of 9.96 U mL −1 . Then, an early efficacy assessment system capable of predicting radiotherapy outcomes within 12 h post‐treatment was constructed, markedly expediting evaluation compared to traditional methods that require weeks. This rapid, precise, and user‐friendly assessment facilitates timely optimization of therapeutic regimens to enhance efficacy while minimizing side effects. This platform represents a significant advancement in precision oncology by transitioning from qualitative imaging to in situ quantitative biomarker tracking.