Engineered UCNF@SiO2@TAMRA-pep nanoflowers enable ultrasensitive activatable FRET imaging of Cathepsin B in vivo

Cathepsin B (CTSB) is a lysosomal cysteine protease implicated in various pathological processes, including cancer progression, inflammation, and apoptosis. Its overexpression or dysregulation serves as a crucial biomarker for disease diagnosis and prognosis. Current imaging modalities often lack the sensitivity, specificity, or real-time monitoring capabilities required to accurately track CTSB activity in complex biological environments. There is a significant need for highly sensitive, activatable probes that can precisely detect CTSB activity, facilitating early disease detection and dynamic assessment of therapeutic responses.

Researchers engineered UCNF@SiO2@TAMRA-pep nanoflowers, a peptide-functionalized upconversion nanoplatform. This probe was constructed via seed-mediated epitaxial growth of core-double-shell nanoflowers, followed by silica encapsulation and covalent conjugation of a TAMRA-labeled CTSB-cleavable peptide (CLLGPLGDDDK). The probe operates on a FRET mechanism: TAMRA quenches the green upconversion emission in the intact probe, with emission restored upon CTSB-mediated peptide cleavage. The platform was evaluated for CTSB detection in buffer, living cells (HeLa, MDA-MB-231), and tumor-bearing mice. Control experiments utilized heat-inactivated CTSB to confirm enzyme specificity.

The UCNF@SiO2@TAMRA-pep probe demonstrated a rapid, concentration-dependent response to CTSB over 0.01-20 μg mL-1, exhibiting excellent linearity (R2 = 0.991) and an ultrasensitive detection limit of 11.4 ng mL-1. This superior performance is attributed to the synergistic effects of the multidimensional nanoflower morphology and the multilayered core-double-shell architecture, which collectively enhance brightness, FRET efficiency, and peptide loading. Comparative studies across core, core-shell, and core-double-shell nanoflower structures confirmed progressively improved luminescence and FRET performance. Control experiments with heat-inactivated CTSB verified the signal recovery was enzyme-specific. The probe exhibited negligible cytotoxicity and enabled sensitive monitoring of intracellular CTSB during cisplatin-induced apoptosis. Cisplatin-treated HeLa cells showed roughly fourfold higher luminescence recovery than MDA-MB-231 cells, reflecting elevated CTSB activation, and the signal correlated linearly with HeLa cell numbers from 5 × 10^3 to 1 × 10^6. In vivo imaging further demonstrated sensitive CTSB detection in tumor-bearing mice without observable toxicity.