Cancer cell discrimination and dynamic viability monitoring through wash-free bioimaging using AIEgens

Cancer cell discrimination and cellular viability monitoring are closely related to human health. A universal and convenient fluorescence system with a dual function of wide-spectrum cancer cell discrimination and dynamic cellular viability monitoring is desperately needed, and is still extremely challenging. Herein we present a series of aggregation-induced emission luminogens (AIEgens) (denoted as IVP) which can allow accurate discrimination between cancer and normal cells and dynamic monitoring of cellular viability through mitochondria–nucleolus migration. By regulating the lengths and positions of alkyl chains in IVP molecules, we systematically studied the discrimination behavior of these AIEgens between cancer cells and normal cells and further investigated how they can migrate between the mitochondria and nucleolus based on the change of mitochondrial membrane potential (ΔΨm). Using IVP-02 as a model molecule, wash-free bioimaging, excellent two-photon properties, and low cytotoxicity were demonstrated. This present work proves that these designed IVP AIEgens show great potential for cancer identification and metastasis monitoring, as well as activity evaluation and screening of drugs.

S3 cells were incubated with these IVP probes (2 µM) in DMEM at 37 o C for 30 min.
Co-staining experiments: MTDR was dissolved in DMSO at a stock concentration of 0.1 mM. To confirm the location of these IVP molecules in mitochondria in cancer cells, HeLa and A549 cells were firstly incubated with 0.2 µM MTDR for 15 min, then stained with 2 µM IVP molecules for 30 min, respectively.

Fluorescence imaging
The confocal fluorescent images were obtained with Zeiss LSM 800 confocal laser scanning microscope. The co-localization coefficient and mean fluorescence intensity of the images were determined by the software with Zeiss LSM 800 confocal microscope. Two-photon microscopy images were obtained with Olympus FV 1200 laser scanning microscope.

RNA titration
Stock solutions of IVP molecules were initially prepared in the solvent of DMSO with the concentration of 5 mM. Stock solution of RNA was prepared in the concentration of 1 mg/mL in Tris buffer solutions (pH = 7.2). To calculate the molar concentration of the stock solution, RNA solution was diluted to 0.01 ‰ and the absorption spectra were measured. The molar concentration of RNA stock solutions was then calculated with the following Eq. (2) 2 : (2) Where C RNA is the concentration of the stock solution, A 260 is the absorbance at 260 nm.
In RNA titration experiments, suitable amount of the stock solutions of IVP molecules and RNA was added into Tris buffer solutions (pH = 7.2) to obtain different concentrations. Then the absorption spectra were acquired, and the fluorescence spectra were also measured with the spectrofluorimeter with the excitation wavelength of 400 nm.

Molecular simulation
The chemical structures of the fluorescent probes were initially optimized with S4 Then MTT (5 mg/mL in DMEM, 20 µL) was added into each well. After 4 h incubation at 37 °C, 100 µL DMSO was added to dissolve the purple crystals. After 20 min incubation, the optical density readings at 570 nm were taken using a plate reader.

Synthetic details, NMR spectra and HRMS spectra
Since the synthesis routes and experimental procedures of the IVP molecules are very similar. Here we take IVP-02 as an example.
The mixture was stirred at room temperature for 5 h. Then the reaction mixture was poured into petroleum ether, and the orange solid was filtrated. After recrystallization, IVPI-12 was obtained as an orange solid (0.26 g, 65%). Finally, IVP-02 was synthesized. IVPI-2 (0.11 g, 0.3 mmol) was dissolved in acetone (10 mL), and a solution of KPF 6 (0.55 g, 3 mmol) in was added. The mixture was stirred at room temperature for 24 h. Acetone was removed under reduced pressure and the residue was purified by silica gel chromatography using CH 2 Cl 2 /MeOH mixture (8:1, v/v) as eluent to give IVP-02 as an orange red solid (66 mg, 56%). 1       For IVP-06, 1