In vivo imaging of leucine aminopeptidase activity in drug-induced liver injury and liver cancer via a near-infrared fluorescent probe

The upregulation of leucine aminopeptidase in hepatopathy models is imaged in vivo for the first time with a near-infrared fluorescent probe.


Apparatus and reagents
Fluorescence measurements were made on a Hitachi F-4600 spectrophotometer in 10 mm × 10 mm quartz cells (Tokyo, Japan). 1 H-and 13 C-NMR spectra were measured with a Bruker DMX-400 spectrometer. Electrospray ionization (ESI) mass spectra were measured on a Shimadzu LC-MS 2010A instrument (Kyoto, Japan). High resolution electrospray ionization mass spectra (HR-ESI-MS) were recorded on an APEX IV FTMS instrument (Bruker, Daltonics). Absorption spectra were recorded in 1-cm quartz cells with a TU-1900 spectrophotometer (Beijing, China). MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] analysis was made on a microplate reader (Molecular Devices SpectraMax i3). Fluorescence imaging was conducted on an FV 1200-IX83 confocal laser scanning microscope (Olympus). Imaging of histological sections was conducted on a BX43 fluorescence microscope (Olympus). In vivo fluorescence imaging was made on a Kodak In-vivo Imaging System FX Pro.

Synthesis of HCA.
3-Nitrophenol (347 mg, 2.5 mmol) and K 2 CO 3 (345 mg, 2.5 mmol) were dissolved in 15 mL CH 3 CN in a flask, and the mixture was stirred at room temperature under argon atmosphere for 10 min. Then, IR-780 iodide (667 mg, 1 mmol) in CH 3 CN (2 mL) was introduced to the mixture via a syringe and the reaction mixture was stirred at room temperature for 4 h. The solvent was then evaporated under reduced pressure and the precipitate was dissolved in CH 2 Cl 2 , followed by washing with water for three times and drying over Na 2 SO 4 . The residue obtained by evaporation was dispersed in 30 mL CH 3 OH for further use in the next step.

General procedure for spectroscopic detection
where A is the absorbance at the excitation wavelength (A is kept between 0.01 and 0.05), I is the integrated area of emission spectra, and η is the refractive index of the solvent. The subscripts s and x refer to the standard and unknown, respectively.
The stock solution (1.0 mM) of probe HCAL was prepared in deoxygenated DMSO.
In a test tube, 4 mL of PBS (pH 7.4) and 25 μL of 1 mM probe were mixed, followed by addition of LAP solution. The final volume was adjusted to 5 mL with PBS. After incubation at 37 °C for 90 min in a thermostat, the reaction solution was transferred to a quartz cell of 1-cm optical length to measure absorbance or fluorescence with λ ex/em = 670/705 nm. At the same time, solution containing no LAP was prepared and measured under the same conditions for comparison.

Cell incubation and fluorescence imaging
Cells ( For comparison, the pixel intensity at least from five cells in each fluorescence image was measured and averaged in this work. For Ace stimulation, an appropriate concentration of Ace solution was added to the dishes containing adherent cells in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin at 37 °C in a humidified 5% CO 2 incubator, and the cells were incubated for different periods of time.

Cytotoxicity assay
The cytotoxicity of HCAL to HepG2 cells was evaluated using standard MTT assay, as described previously (Song et al, J. Mater. Chem. 2012, 22, 12568).

In vivo imaging of mice
Ace was dissolved in warm saline (

Western blot analysis
HepG2 cells were lysed with RIPA buffer, and the cell lysates were diluted with PBS to obtain a solution of about 0.5 mg/mL of total proteins. The protein standard solution of 0.5 mg/mL was prepared. The protein concentration and western blot analyses were made following the previous method (Gong et al. Chem. Sci. 2016, 7, 788-792).