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

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.