溶酶体靶向远红光至近红外荧光探针用于监测铁死亡过程中粘度的变化

doi:10.6023/cjoc202203039

摘要/Abstract

阐明病理过程与溶酶体粘度之间的内在关系是一个巨大的挑战. 到目前为止, 还未有文献报道铁死亡过程中溶酶体粘度的变化情况. 在本工作中, 设计并合成了一种基于氟硼二吡咯(BODIPY)染料可用于检测粘度的新型远红光至近红外荧光探针FNVP. 该探针对粘度变化具有良好的响应能力(λ_ex=600 nm, λ_em=680 nm). 在磷酸盐缓冲生理盐水(PBS)-甘油体系(1~219 cP)中, 探针FNVP在680 nm处的荧光强度约增加50倍. 其次, 探针具有专一性好、光稳定性强、细胞毒性小等优点, 并可用于溶酶体定位及制霉菌素诱导下的细胞粘度监测. 最后, 利用共聚焦荧光技术, 在细胞水平上进行铁死亡过程中的溶酶体粘度成像. 这项工作的实施将帮助我们进一步了解铁死亡过程的生物学效应.

关键词: 粘度, 荧光探针, 溶酶体, 近红外, 铁死亡

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

引用此文

赖燕琴, 陈雪, 陈芳, 倪林晨, 王婷, 祝子坪, 满菊, 姜春筱, 谢振达. 溶酶体靶向远红光至近红外荧光探针用于监测铁死亡过程中粘度的变化[J]. 有机化学, 2022, 42(9): 2850-2856.

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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图/表 8

图式1. 探针FNVP对粘度的响应机理

Scheme 1. Response mechanism of probe FNVP to viscosity

图式2. 探针FNVP的合成路线

Scheme 2. Synthesis route of probe FNVP

图1. (a)荧光探针FNVP (10 μmol/L)在不同比例PBS(P)-甘油(G)混合溶液的荧光光谱; (b) PBS-甘油溶剂中log I680 nm和log η之间的线性关系(λex=600 nm, λem=680 nm)

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)

图2. 在10 mmol/L PBS缓冲液中探针FNVP (10 μmol/L)在不同物种(500 μmol/L)的荧光强度

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

图3. 在不同pH的PBS-甘油混合溶液(V∶V=3∶7, 10 mmol/ L PBS)中探针FNVP (10 μmol/L)的荧光强度变化(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)

图4. Hela细胞在探针FNVP (10 μmol/L)分别和Lyso-Tracker Green (0.5 μmol/L)、Mito-Tracker Green (0.5 μmol/L)、Hoechst (0.5 μmol/L)共孵化下的共定位成像

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

图5. 探针FNVP在HeLa细胞中的共聚焦荧光图像

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

图6. 监测Erastin诱导铁死亡过程中HeLa细胞粘度变化和HeLa细胞在不同条件下的荧光图像

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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