Reaction-free and MMP-independent fluorescent probes for long-term mitochondria visualization and tracking

We present two mitochondria-immobilized fluorescent probes ECPI-12 and IVPI-12 for long-term mitochondria visualization and tracking regardless of MMP changes.


Apparatus and general methods
All the chemicals were purchased and used as received without further purification unless otherwise specified. The mitochondrial probe MitoTracker Deep Red FM (MTDR), lysosomal probe LysoTracker Deep Red (LTDR) were purchased from Molecular Probes. The UV-visible-near-IR absorption spectra of dilute solutions were recorded on a HITACH U-2910 spectrophotometer using a quartz cuvette with 1 cm path length. One-photon spectra were obtained on a HITACH F-2700 spectrafluorimeter equipped with a 450-W Xe lamp. Two-photon excited fluorescence (TPEF) spectra were measured on a SpectroPro300i, and the pump laser beam came from a mode-locked Ti: sapphire laser system with a pulse duration of 160 fs and a repetition rate of 76 MHz.

Measurement of fluorescence quantum yield and two-photon absorption cross section
Fluorescence quantum yield (Φ) can be calculated by means of Eq. (1): [1] (1) s and r refer to the sample and the reference materials, respectively. Φ is the fluorescence quantum yield, F is the integrated emission intensity, A stands for the absorbance, and n is the refractive index. In this work, the quantum yields were calculated by using fluorescein (Φ = 0.95, pH = 13) as the standard. [2] Two-photon absorption cross-section (δ) was measured using the two-photon induced fluorescence method, and thus the δ can be calculated by means of Eq. (2): [1] (2) = Φ Φ F is TPEF integral intensity. Φ is the fluorescence quantum yield. δ r is the two-photon absorption cross-section of fluorescein in sodium hydroxide aqueous solution (pH = 13.0). [3] 1.3 Cell culture and staining, and tissue staining S3 Cell culture: HeLa and A549 cells were grown in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin in a 5% CO 2 incubator at 37 o C.
Cell staining experiment: ECPI-12 and IVPI-12 was dissolved in DMSO at a stock concentration of 0.5 mM, respectively. HeLa and A549 cells were placed on glass coverslips and allowed to adhere for 48 h. HeLa and A549 cells were incubated with probes in DMEM for 30 min at 37 o C. Mitophagy tracking: We monitored the co-localization coefficient values of ECPI-12/IVPI-12 and LTDR during mitophagy process. HeLa cells were stained with 2 µM ECPI-12/IVPI-12 and 0.2 µM LTDR, and then treated with 10 μM CCCP and 7.5 μM pepstatin A to induce mitophagy. We recorded the fluorescent images at different treated time points of 0 h, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, and obtained the corresponding co-localization coefficient.
Tissue staining: The rat skeletal muscle tissues were directly removed from just killed adult wistar rat (purchased from Laboratory Animal Center, Shandong University). Then the tissues were stained with ECPI-12/IVPI-12 (0.2 µM) at room temperature in H-DMEM supplemented with 10 % fetal bovine serum (FBS) and 1% penicillin and streptomycin for 1 h. The tissues were washed with PBS to remove the unbound probe before performing two-photon imaging.

Cell-viability assay
The study of the effect of ECPI-12/IVPI-12 on cell viability was carried out using the standard MTT assay. HeLa cells growing in log phase were seeded into 96-well plates (ca. 1 × 10 4 cells/well) and allowed to adhere for 24 h. ECPI-12/IVPI-12 and MTDR dissolved in DMEM at concentrations of 0.1 µM, 0.2 µM, 0.5 µM, and 1.0 µM, respectively, were added into the wells as the treatment group (200 µL/well), and DMEM without dyes was added into the wells as the negative control group. The cells were incubated for 48 h at 37 °C under 5% CO 2 . Then MTT (5 mg/mL in DMEM) was added into each well. After 4 h incubation at 37 °C, 200 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. Cytotoxic experiment was repeated for three times.
Synthesis of compound 1: KOH (8.4 g, 150 mmol) was dissolved in N,N-dimethylformamide (DMF) (30 mL) and the solution was stirred at room temperature for 30 min. The DMF containing carbazole (5.0 g, 29.8 mmol) was then added and reacted for 1 h. Then 2-Bromoethyl ethyl ether (6.8 g, 45 mmol) was added dropwise into the above solution and the mixture reacted overnight. The reaction mixture was poured into water, and the yellow solid was filtrated. After recrystallization, compound 1 was obtained as a white solid (6.4 g, 90%). 1

Crystallographic data
Table S1 Crystal data and structure refinement for ECPI-12.

Table S2
Crystal data and structure refinement for IVPI-12. λ abs max and λ em max are the maximum absorption and one-photon fluorescence wavelengths, respectively. ε is molar extinction coefficient. Φ is one-photon fluorescence quantum yield determined by using fluorescein (Φ = 0.95) as the standard. Concentration: 10 µM.