Near-Infrared Fluorescent Probe with Remarkable Large Stokes Shift and Favorable Water Solubility for Real-Time Tracking Leucine Aminopeptidase in Living Cells and In Vivo

• Published in: Analytical chemistry (Washington) 2017-11, Vol.89 (22), p.12319-12326
• Main Authors: Zhang, Wenda, Liu, Feiyan, Zhang, Chao, Luo, Jian-Guang, Luo, Jun, Yu, Wenying, Kong, Lingyi
• Format: Article
• Language: English
• Subjects: Aminopeptidase; Animal models; Biocompatibility; Cancer; Cell membranes; Cells; Diagnostic software; Diagnostic systems; Enzymatic activity; Enzymes; Fluorescence; Fluorescent indicators; I.R. radiation; In vivo methods and tests; Infrared radiation; Invasiveness; L-leucine; Leucine; Liver; Liver cancer; Medical imaging; Membrane permeability; Near infrared radiation; Pathogenesis; Permeability; Proteolysis; Proteolytic enzymes; Real time; Selectivity; Sensitivity; siRNA; Solubility; Toxicity; Tracking; Water
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.7b03332

Leucine aminopeptidase (LAP) is a kind of proteolytic enzymes and associated closely with pathogenesis of cancer and liver injury. Accurate detection of LAP activity with high sensitivity and selectivity is imperative to detect its distribution and dynamic changes for understanding LAP’s function and early diagnosing the disease states. However, fluorescent detection of LAP in living systems is challenging. To date, rarely fluorescent probes have been reported for imaging LAP in vivo. In this study, a novel probe (TMN-Leu) was developed by conjugating a near-infrared dicyanoisophorone derivative fluorophore with LAP activatable l-leucine amide moiety for the first time. TMN-Leu featured large Stokes shift (198 nm), favorable water solubility, ultrasensitive sensitivity (detection limit of ∼0.38 ng/mL), good specificity, excellent cell membrane permeability, low toxicity, and a prominent near-infrared emission (658 nm) in response to LAP. TMN-Leu has been successfully applied to track LAP of cancer cells and normal cells, monitor LAP changes in different disease models, and rapidly evaluate LAP inhibitor in cell-based assay. Notably, this probe firstly revealed that HCT116 cells with higher LAP activity were more invasive than LAP siRNA transfected HCT116 cells, suggesting that LAP might serve as an indicator reflecting the intrinsic invasion ability of cancer cells. Finally, TMN-Leu was also employed for in vivo real-time imaging LAP in living tumor-bearing nude mice with low background interference. All together, our probe possesses potential value as a promising tool for diagnostic application, cell-based screening inhibitors and in vivo real-time tracking enzymatic activity in preclinical applications.