Imaging of intracellular sulfane sulfur expression changes under hypoxia stress via a selenium-containing near-infrared fluorescent probe
Min Gao【高敏】,†a,c Rui Wang【王锐】,†a,b Fabiao Yu【于法标】,a,b* Bowei Li【李博伟】a and Lingxin Chen【陈令新】a,d*

†Min Gao and Rui Wang contributed equally.

^a Key Laboratory of Coastal Environmental Processes and Ecological Remediation, Research Centre for Coastal Environmental Engineering and Technology, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China.

^b Institute of Functional Materials and Molecular Imaging, College of Emergency and Trauma, Hainan Medical University, Haikou 571199, China

c University of Chinese Academy of Sciences, Beijing 100049, China.

Abstract

Hypoxia is a significant global issue affecting the health of organism. Homeostasis of oxygen is critical for mammalian cell survival and cellular activities. Hypoxia stress can lead to cell injury and death, which attributes to many diseases. Sulfane sulfur is involved in many crucial roles of physiological processes via maintaining intracellular redox state and ameliorating oxidative damage. Therefore, real-time imaging of sulfane sulfur changes is important for understanding their biofunction in cells. In this work, we develop a new near-infrared (NIR) fluorescent probe BD-diSeH for imaging of sulfane sulfur changes in cells and in vivo under hypoxia stress. The probe includes two moieties: the NIR azo-BODIPY fluorophore equipped with a strong nucleophilic phenylselenol group (-SeH). The probe is capable for tracing the dynamic changes of endogenous sulfane sulfur based on a fast and spontaneous intramolecular cyclization reaction. The probe has been successfully used for imaging sulfane sulfur in 3D-multicellular spheroid and in mice hippocampus under hypoxia stress. The overall levels of sulfane sulfur are affected by the degree and time of hypoxia stress. The results reveal a close relationship between sulfane sulfur and hypoxia in living cells and in vivo, which is better understanding the physiological and pathological processes involved by sulfane sulfur. Moreover, to indicate the effects of environmental hypoxia on aquatic animals, this excellent probe has been applied for sulfane sulfur detection in hypoxic zebrafish.

Scheme 2. Design strategy and proposed detection mechanism of BD-diSeH towards sulfane sulfur.

Figure 1. (a) UV-vis absorption spectra of BD-diSeH (10 μM) before and after treatment of Na₂S₄ (20 μM). (b) Fluorescence spectra of BD-diSeH (10 μM) upon addition of Na₂S₄ (0-20 μM). Spectra were obtained after incubation of the probe with Na₂S₄ for 5 min. (c) The corresponding linear relationship between fluorescent intensity and Na₂S₄ concentration (0-20 μM) in buffer solution. The red point was the mean fluorescent intensity of mice serum. (d) Time-dependent enhancement in fluorescence response of BD-diSeH (10 μM) toward various RSS. 1. Na₂S₂ 20 μM; 2. Na₂S₄ 20 μM; 3. PhCH₂S₄CH₂Ph 20 μM; 4. Cys-Poly-Sulfide 20 μM; 5. S₈ 20 μM; 6. NaHS 100 μM, 7. GSH 1 mM, 8. Cys 500 μM, 9. Hcys 500 μM, 10. Cys-Cys 500 μM, 11. GSSG 500 μM. Bars represent fluorescent intensity during 0, 1, 2, 3, and 4 min after addition of various RSS. Time-dependent fluorescence changes of BD-diSeH upon addition of Na₂S₄ (20 μM) in the absence/presence of interfering thiols H₂S, GSH and Cys (1 mM, e and f). All spectra were acquired in 10 mM HEPES (20% fetal bovine serum, v/v, pH 7.4). λ_ex/em= 707/737 nm.

Figure 4. Fluorescence images of A549 cells using BD-diSeH at different level of oxygen concentration (20%, 10%, 5%, 1%, 0.1%). A549 cells were incubated with BD-diSeH (1 μM) for 3 h under various oxygen concentrations (a). Then the cells were further incubated with Calcein-AM (5 μM, b), and Hoechst 33342 (1 μg/mL, c) for 30 min. (d) Colocalization images of the red, green and blue channels. (e) Correlation plot of the red and green channels. (f) Flow cytometry assay of A549 cells using BD-diSeH at different level of oxygen concentration (20%, 10%, 5%, 1%, 0.1%). Fluorescence collection windows constructed from 680 to 780 nm for BD-diSeH, from 500 to 550 nm for Calcein-AM, from 425 to 500 nm for Hoechst 33342, λ_ex = 635, 488, and 405 nm, respectively.

Figure 5. (a) Fluorescence images of SH-SY5Y MCs upon incubation with BD-diSeH (1 μM) for 8 h at 37 ℃. The representative images were acquired using confocal microscopy with Z-stack imaging at 10 μm intervals. Fluorescence collection windows were constructed from 680-780 nm. (b) Bright field image and overlay image. (c) The signal intensity profile of selected cross-section of the MCs in Figure 5b (yellow arrow). (d) Perspective observation of MCs with Z-stack reconstruction.

Figure 6. (a) Confocal fluorescence images of hippocampus slice using BD-diSeH. The hippocampus slices were incubated with BD-diSeH (1 μM) for 20 min. λ_ex =635 nm. Fluorescence collection windows was constructed from 680-780 nm. (b) Normalized fluorescent intensity of Figure 6a. Data are presented as means ± SD (n = 5). (c) Western blot analysis of HIF-1α. β-actin was taken as the loading control. (d) Fluorescence images of BALB/c mice visualizing sulfane sulfur level changes using BD-diSeH. Images displayed represent emission intensities collected window: 700−800 nm, λ_ex = 680 nm. Group a was injected i. p. with BD-diSeH (10 μM, 50 μL in 1:9 DMSO-saline, v/v) for 20 min. Group b was firstly pretreated with Na₂S₄ (20 μM, 50 μL in saline), then injected with BD-diSeH for 20 min. Group c was firstly injected with LPS (10 μg/mL, 100 μL in 1:9 DMSO-saline, v/v) for 24 h, and PLP (1 μM, 50 μL in saline) for 2 h, then injected with BD-diSeH for 20 min. (e) Mean fluorescent intensity of Group a−c. The total number of photons from the entire peritoneal cavity of the mice was integrated. Data are presented as means ± SD (n = 5).

http://pubs.rsc.org/en/content/articlelanding/2018/tb/c8tb01794h#!divAbstract

The article was received on 10 Jul 2018, accepted on 19 Sep 2018 and first published on 20 Sep 2018

DOI:10.1039/C8TB01794H

Citation: J. Mater. Chem. B, 2018, Accepted Manuscript 

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