Lysosome-Targeting Fluorogenic Probe for Cathepsin B Imaging in Living Cells

Abstract

Cathepsin B (CTB) is a lysosomal protease which has been recognized as a promising biomarker for many malignant tumors, and accurate detection of its activity is important in early diagnosis of cancers and predicting metastasis. Herein, we reported a lysosome-targeting fluorogenic small-molecule probe for fluorescence imaging of lysosomal CTB in living cancer cells by incorporating a CTB-recognitive peptide substrate Cbz-Lys-Lys-p-aminobenzyl alcohol (Cbz-Lys-Lys-PABA) and a lysosome locating group morpholine. We demonstrated that the probe could be efficiently activated by CTB to generate ∼73-fold enhancement in fluorescence under acidic lysosomal environment (pH 4.5-6.0), allowing for high sensitivity and specificity to detect CTB. Fluorescence imaging results showed selective accumulation and fluorescence turn-on in the lysosomes of cancer cells, which were capable of reporting on lysosomal CTB activity in cancer cells and normal tissue cells. This study highlights the potential of using a lysosome-targeting group to design a sensitive and specific fluorogenic probe for fluorescence imaging of lysosomal CTB in living cells.

Unprecedented Sensitivity in a Probe for Monitoring Cathepsin B: Chemiluminescence Microscopy Cell‐Imaging of a Natively Expressed Enzyme

“…No chemiluminescence signal was observed from the 3T3 fibroblast cells. These observations correlate with the fact that RAW 246.7 and CT26 cells overexpress cathepsin B, whereas the mouse fibroblast 3T3 cells, as the normal tissue, have significantly lower levels of cathepsin B . These results suggest that probe 4 is able to differentiate cancerous cells from normal tissue.…”

Unprecedented Sensitivity in a Probe for Monitoring Cathepsin B: Chemiluminescence Microscopy Cell‐Imaging of a Natively Expressed Enzyme

“…Therefore,R AW 264.7 (leukemia), CT26 (colon cancer), and 3T3 fibroblast cells (as acontrol for normal cells) were incubated with probe 3 or 4 and imaged using an LV 200 Olympus microscope.A ss hown in Figure 6, no light emission was observed when cells were incubated with probe 3.I ncubation of probe 4 with either the RAW 264.7 or the CT26 cells resulted in bright chemiluminescence cell images.Nochemiluminescence signal was observed from the 3T3 fibroblast cells.These observations correlate with the fact that RAW246.7 and CT26 cells overexpress cathepsin B, whereas the mouse fibroblast 3T3 cells,a st he normal tissue, have significantly lower levels of cathepsin B. [35][36][37] These results suggest that probe 4 is able to differentiate cancerous cells from normal tissue.P robe 3 most likely failed as ac ell imaging agent for cathepsin Bbecause of insufficient solubil-…”

Firefly Luciferin‐Inspired Biocompatible Chemistry for Protein Labeling and In Vivo Imaging

“…In addition to bioluminescence, the change of fluorescenceg enerating from the fast reaction betweenC BT andi ntracellular l-Cysc ould also be readily appliedt ob uild fluorogenic probesf or cell imaging. [128][129][130] Another advantage of the reaction is the formation of condensation product with as ingle stereo/regioisomer, which allows for precise control of DNA modifications [131] or site-specific immobilization of biomolecules. [132] Other applications such as controlling of drug delivery for overcoming multidrug resistance have been demonstrated as well.…”