A highly selective near-infrared fluorescent probe for imaging H2Se in living cells and in vivo

Abstract

Hydrogen selenide (H₂Se), a highly reactive Se species, is an important selenium metabolism intermediate involved in many physiological and pathological processes. This compound is of scientific interest with regard to the real-time monitoring of H₂Se in living cells and in vivo to understand the anti-cancer mechanism of selenium. However, monitoring H₂Se in living cells is still challenging due to the lack of straight forward, highly selective and rapid methods. Here, we developed a novel small-molecule fluorescent probe, NIR-H₂Se, for imaging endogenous H₂Se. NIR-H₂Se exhibited high selectivity toward H₂Se over selenocysteine (Sec), H₂S and small molecule thiols and was successfully used to image the H₂Se content in HepG2 cells during Na₂SeO₃-induced apoptosis. Increased H₂Se content and reduced ROS levels were observed under hypoxic conditions compared to normoxic conditions, which indicated that the cell apoptosis induced by Na₂SeO₃ under a hypoxic environment is via a non-oxidative stress mechanism. Thus, this probe should serve as a powerful tool for exploring the physiological function of H₂Se and Se anticancer mechanisms in a variety of physiological and pathological contexts.

Scheme 1. Synthesis of NIR-H₂Se. Reaction conditions: (a) NaH, DMF, rt, 73%; (b) SnCl₂, HCl, CH₃OH, 70 °C, 45%; (c) SeO₂, grind, 71%.

Scheme 2. Proposed mechanism for the selective reaction of H₂Se.

Fig. 1. (a) Fluorescence response of 10 μM NIR-H₂Se to differing amounts of H₂Se. (b) Linear correlation between the emission intensity and H₂Se concentration. All spectra were acquired in 10 mM PBS with a pH of 7.4 (λ _ex/λ _em = 688/735 nm). The data were expressed as the mean ± standard deviation (SD) across three experiments.

Fig. 2. (a) Fluorescence intensity changes for NIR-H₂Se (10 μM) after adding 100 equiv. of thiols, Na₂S, Na₂SeO₃, Sec, NAC, TrxR, Vc, various biologically related ROS and 10 equiv. of NO. The black bars show the addition of one of these interferents to a 10 μM NIR-H₂Se solution. The red bars represent the addition of both H₂Se and one interferent to the probe solution. (b) Time course for the fluorescence intensity of 10 μM NIR-H₂Se with 10 μM H₂Se in a 10 mM PBS, pH = 7.4, at room temperature.

Fig. 3. Confocal fluorescence imaging of endogenous H₂Se in HepG2 cells treated with sodium selenite under normoxic (20% pO₂) and hypoxic (1% pO₂) conditions. (a) The fluorescence changes for the HepG2 cells exposed to different sodium selenite concentrations (2–10 μM) for 12 h and incubated with NIR-H₂Se (10 μM). (b) The fluorescence changes of the HepG2 cells exposed to 5 μM of sodium selenite for different times (0–12 h) and incubated with NIR-H₂Se (10 μM). The fluorescence was imaged using a confocal microscope with 633 nm excitation and 650–750 nm collection. (c) The fluorescence intensity for (a). (d) The fluorescence intensity for (b). The data were normalized to the control, and statistical analyses were performed using a two-tailed Student's t-test (n ≥ 4 fields of cells) relative to the control. *P < 0.01 and the error bars are ± the standard error measurement.

Fig. 4. Confocal fluorescence imaging of H₂O₂ in HepG2 cells treated with sodium selenite under normoxic (20% pO₂) and hypoxic (1% pO₂) environments. (a) The fluorescence changes in the HepG2 cells exposed to different sodium selenite concentrations (2–10 μM) for 12 h and incubated with a H₂O₂ probe (10 μM). (b) The fluorescence changes for the HepG2 cells exposed to a 5 μM sodium selenite solution for different times (0–12 h) and incubated with a H₂O₂ probe (10 μM). The fluorescence was imaged using a confocal microscope with 532 nm excitation and 600–700 nm collection. (c) The fluorescence intensity for (a). (d) The fluorescence intensity for (b). The data were normalized to the control, and statistical analyses were performed using a two-tailed Student's t-test (n ≥ 4 fields of cells) relative to the control. *P < 0.01 and the error bars are ± the standard error measurement.

Fig. 5. (a) In vivo fluorescence imaging of H22 tumor-bearing mice injected with the probe NIR-H₂Se (10 μM) in response to sodium selenite (10 μM) at different times. (b) The fluorescence intensity for (a). The data were normalized to the control.