A Lysosome-Targeted Far-Red to Near-Infrared Fluorescent Probe for Monitoring Viscosity Change During the Ferroptosis Process

doi:10.6023/cjoc202203039

Abstract

Elucidating the intrinsic complex relationship between pathophysiological processes and lysosomal viscosity is a tremendous challenge. So far, the change of lysosomal viscosity during the ferroptosis process has not been reported. In this study, a novel far-red to near-infrared viscosity fluorescent probe (FNVP) was designed and synthesized based on boron dipyrromethene (BODIPY) dye. The probe exhibited excellent responsiveness to viscosity change (λ_ex=600 nm, λ_em=680 nm). In the phosphate buffered saline (PBS)-glycerol system (1~219 cP), the emission intensity of probe FNVP at 680 nm had about a 50-times increase. Furthermore, the probe showed the advantages of high selectivity, remarkable photostability and negligible cytotoxicity. Probe FNVP was lysosome-targetable and could be used for monitoring lysosomal viscosity changes induced by nystatin. Finally, using confocal fluorescence microscopy, lysosome viscosity was imaged during the ferroptosis process at the cellular level. All those will be beneficial to further comprehending the biological effectiveness of ferroptosis.

Key words: viscosity, fluorescent probe, lysosome, near-infrared, ferroptosis

Cite this article

Yanqin Lai, Xue Chen, Fang Chen, Linchen Ni, Ting Wang, Ziping Zhu, Ju Man, Chunxiao Jiang, Zhenda Xie. A Lysosome-Targeted Far-Red to Near-Infrared Fluorescent Probe for Monitoring Viscosity Change During the Ferroptosis Process[J]. Chinese Journal of Organic Chemistry, 2022, 42(9): 2850-2856.

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Fig. & Tab. 8

Scheme 1. Response mechanism of probe FNVP to viscosity

Scheme 2. Synthesis route of probe FNVP

Figure 1. (a) Fluorescence spectra of probe FNVP (10 μmol/L) in different ratios of PBS(P)-glycerol (G) solution; (b) the linear relationship of the log I680 nm versus log η plot in the PBS/glycerol solvents (λex=600 nm, λem=680 nm)

Figure 2. Fluorescence intensity of probe FNVP (10 μmol/L) toward various species (500 μmol/L) in 10 mmol/L PBS buffer (1) Blank, (2) ClO–, (3) Ca2+, (4) Zn2+, (5) Fe3+, (6) Al3+, (7) Cu2+, (8) Mg2+, (9) Na+, (10) glucose, (11) GSH, (12) Hcy, (13) Cys, (14) NaHS, (15) tert-butyl hydroperoxide, (16) H2O2, (17) $\text{HSO}_{3}^{-}$, (18) HNO, (19) 90% (volume ratio) glycerol. λex=600 nm, λem=680 nm

Figure 3. Fluorescence intensity of probe FNVP (10 μmol/L) at different pH values in PBS/glycerol system (V∶V=3∶7, 10 mmol/L PBS) (λex=600 nm, λem=680 nm)

Figure 4. Colocalization images of living HeLa cells co-treated with probe FNVP (10 μmol/L) and Lyso-Tracker Green (0.5 μmol/L), Mito-Tracker Green (0.5 μmol/L), and Hoechst (0.5 μmol/L), respectively (a1) Fluorescence image of Lyso-Tracker Green: λex=488 nm, λem=520~590 nm; (a2) fluorescence image of probe FNVP: λex=552 nm, λem=630~700 nm; (a3) the merged image of (a1), (a2) and bright field. Insert: intensity scatter plot of probe FNVP and Lyso-Tracker Green. (b1) Fluorescence image of Mito-Tracker Green: λex=488 nm, λem=520~590 nm; (b2) fluorescence image of probe FNVP: λex=552 nm, λem=630~700 nm; (b3) the merged image of (b1), (b2) and bright field. Insert: intensity scatter plot of probe FNVP and Mito-Tracker Green. (c1) Fluorescence image of Hoechst: λex=405 nm, λem=430~490 nm; (c2) fluorescence image of probe FNVP: λex=552 nm, λem=630~700 nm; (c3) the merged image of (c1), (c2) and bright field. Scale bar: 20 μm

Figure 5. Confocal fluorescence images of probe FNVP in HeLa cells The cells were pretreated with (a~c) none and (d~f) nystatin (20 μmol/L) for 30 min and then treated with probe FNVP (10 μmol/L) for another 30 min. λex=552 nm, λem=630~700 nm. Scale bar: 20 μm

Figure 6. Monitoring of viscosity change in ferroptosis of HeLa cells induced by Erastin and fluorescence images of HeLa cells under different conditions (a1~a3) cells only; cells pretreated with Erastin (10 μmol/L) for (b1~b3) 0 h, (c1~c3) 4 h, or (d1~d3) 8 h and then incubated with probe FNVP (10 μmol/L) for 30 min. (e1~e3) cells pretreated with Erastin (10 μmol/L) in the presence of DFO (100 μmol/L) for 8 h and then incubated with probe FNVP (10 μmol/L) for 30 min. λex=552 nm, λem=630~700 nm. Scale bar: 20 μm

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