Negatively Charged Peptides-Assisted Self-Assembled Mitochondria-Targeting Nanoprobe for Detecting Enterokinase and...

Surgical resection remains the primary treatment in human breast cancer. However, the accurate and rapid delineation of tumor margins intraoperatively is still a significant challenge. Herein, we designed a near-infrared fluorescent probe DCIP-ENTK by incorporating dicyanoisophorone dye with a negatively charged peptide, which can be specifically cleaved by enterokinase (ENTK, a tumor biomarker for breast cancer). In vitro experiments confirmed that the probe could quickly (<10 min), specifically, and sensitively (LOD = 0.031 U/mL) respond to ENTK. Notably, this probe was engineered as a peptide-based architecture that can self-assemble into a nanoprobe. This design not only provides excellent biocompatibility and in vivo stability, but also enables specific mitochondria-targeting within cancer cells. Upon cleavage by ENTK, the released hydrophobic fluorophores aggregated at the enzyme-rich site. This unique aggregation-induced retention effect enabled long-term, high-contrast in situ imaging of tumor margins in tumor-bearing mice and human breast cancer samples, which offers a powerful and clinically translatable tool for improving the precision of cancer surgery. Finally, we also achieved the quantitation of ENTK levels in artificial human duodenal juice with recovery rates from 93.5% to 99.8%. These results confirm the suitability of our probe for the quantitation of ENTK in real samples.