Point-of-care Ratiometric Fluorescence Imaging of Tissue for the Diagnosis of Ovarian Cancer

During a minimally invasive tumor resection procedure, it is still a challenge to rapidly and accurately trace tiny malignant tumors in real time. Fluorescent molecular imaging is considered an efficient method of localizing tumors during surgery due to its high sensitivity and biosafety. On the basis of the fact that γ-glutamyltranspeptidase (GGT) is overexpressed in ovarian cancer, we herein designed a highly sensitive ratiometric fluorescent GGT-responsive probe Py-GSH for rapid tumor detection. Methods: The GGT response probe (Py-GSH) was constructed by using GSH group as a response group and pyrionin B as a fluorescent reporter. Py-GSH was characterized for photophysical properties, response speed and selectivity of GGT and response mechanism. The anti-interference ability of ratiometric probe Py-GSH to probe concentration and excitation power was evaluated both in vitro and in tissue. The biocompatibility and toxicity of the ratiometric probe was examined using cytoxicity test. The GGT levels in different lines of cells were determined by ratiometric fluorescence imaging and cytometry analysis. Results: The obtained probe capable to rapidly monitored GGT activity in aqueous solution with 170-fold ratio change. By ratiometric fluorescence imaging, the probe Py-GSH was also successfully used to detect high GGT activity in solid tumor tissues and small peritoneal metastatic tumors (~1 mm in diameter) in a mouse model. In particular, this probe was further used to determine whether the tissue margin following clinical ovarian cancer surgery contained tumor. Conclusion: In combination of ratiometric fluorescence probes with imaging instrument, a point-of-care imaging method was developed and may be used for surgical navigation and rapid diagnosis of tumor tissue during clinical tumor resection.


Supplemental Experimental Procedures
Theoretical calculations. The structure optimization of compound was performed with the Gaussian 03 package using B3LYP density functional theory (DFT). The 6-31G(d) basis set was used to treat all atoms. The contours of the molecular orbitals were plotted. On the basis of ground-and excited-state optimization, the time-dependent density functional theory (TDDFT) approach was applied to predict their absorption and emission properties.
The solvent effect (CH 2 Cl 2 ) was simulated using the polarizable continuum model (PCM) in which the solvent cavity is regarded as a union of interlocking atomic spheres.
Cell culture. The cell lines SKOV3, CAOV3 and HOSEpiC were provided by the Institute of Biochemistry and Cell Biology, SIBS, CAS (China). The cells were grown in DMEM (modified Eagle's medium) supplemented with 10% FBS (fetal bovine serum) at 37 ºC and 5% CO 2 . All cells were planted on 14 mm glass coverslips and keep to adhere for 24 h Cytotoxicity test. The in vitro cytotoxicity was measured using a standard methyl thiazolyl tetrazolium (MTT, Sigma Aldrich) assay in SKOV3, CAOV3 and HOSEpiC cell lines.
Briefly, cells growing in log phase were seeded into 96-well cell culture plate at 1×10 4 /well.
Py-GSH was added to the wells of the treatment group at concentrations of 2, 5, 10, 25, 50 μM/mL. For the negative control group, 1 μL/well solvent was diluted in DMEM with the final concentration of 1 %. The cells were incubated for 24 h at 37 °C under 5 % CO 2 . The combined MTT/PBS solution was added to each well of the 96-well assay plate and incubated for an additional 4 h. After removal of the culture solution, 200 μL DMSO was added to each well, shaking for 10 min at shaking table. An enzyme-linked immunosorbent assay (ELISA) reader was used to measure the OD570 (absorbance value) of each well referenced at 490 nm.
The following formula was used to calculate the viability of cell growth: Viability (%) = (mean of absorbance value of treatment group / mean of absorbance value of control) × 100 Synthetic routine of the Py-GSH [1,2] Scheme S1. Synthetic routine of the Py-GSH.
Compound S1. 2.00 g (3.8 mmol) Pyronin B was suspended in methanol (300 mL). The solution was heated to 60 °C for 30 min. To the solution was slowly added 6 x 300 mg (6 x 6 mmol, 6 x 1 equiv.) sodium borohydride over the course of 15 minutes. Following stirred the solutions for another 30 minutes, cooled to room temperature, then evaporated all of the solvent. The violet residue was taken up in 100 mL water and 100 mL dichloromethane and the organic layer collected. The aqueous layer was further extracted with 2 x 150 mL DCM, dried over Na 2 SO 4 , and concentrated to yield (0.93 g, 1.9 mmol, 47%) S1 as a magenta solid and used without further purification to the next step.
Compound S2. 0.5 g (1.05 mmol) S1 was dissolved in 25 mL acetone. The solution purged with Ar, and cooled to 0 °C. To the solution was added 3 x 100 mg potassium permanganate over 30 minutes. After 15 minutes, TLC analysis showed full consumption of starting material.
According to the orbital distributions, no evident charge transfer was observed. We studied the dependence of both the absorption spectra and emission property of Py-GSH on the solvent ( Figure S2). There was no evident change in the absorption spectra a nd emission property in different solvents, which demonstrated that the optical property of Py-GSH was attributed to the transition of π-π * . According to the same calculation method, the lowest excited state of amino-modified Py-CG was assigned to HOMO -LUMO, and the two orbitals were still mainly distributed over the entire conjugated backbone. However, an evident change in charge distribution on the meso-N of amino-8 modified Py-CG from HOMO to LUMO was obtained. These findings indicated that the S N Ar substitution-rearrangement reaction resulted in a transfer of the transition method of the excited state, which was responsible for the change in optical properties from Py-GSH to amino-modified Py-CG.         with 10 μM Py-GSH saline for 10 min. In fluorescence imaging, the emission channel at 560±15 nm (Green channel) and 650±15 nm (Red channel) were collected. In ratiometric imaging, the ratio of emission intensity at 560±15 nm to that at 650±15 nm was chosen as the detected signal. Ex=490 nm.  and 650±15 nm (Red channel) were collected. The ratio signal was calculated from the emission intensity at 560±15 nm to that at 650±15 nm. collected. In ratiometric imaging, the ratio of emission intensity at 560±15 nm to that at 650±15 nm was chosen as the detected signal. Ex = 490 nm. Scale bar, 2 mm.