A lysosomal targeted fluorescent probe based on coumarin for monitoring hydrazine in living cells with high performance

Abstract

A lysosomal targeted fluorescent probe based on coumarin for monitoring hydrazine (N₂H₄) in living cells was designed and synthesised. The novel fluorescent probe Cou-Lyso-N₂H₄, in response to N₂H₄, exhibited good selectivity, low cytotoxicity, and lysosomal localization, which could be suitable for studying the harmfulness of N₂H₄ in subcellular organelles during various physiological processes.

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Hydrazine (N₂H₄) possesses strong reducibility and highly reactive alkalinity due to the α-effect,¹ and is widely used as missile and rocket fuel,² reducing agent,³ polymer cross-linking agent,⁴ and raw material in medicine.⁵ On the other hand, hydrazine also shows good water solubility and high toxicity, however, excessive inhalation would bring a serious threat to human health and the environment. According to previous literatures, when people are often exposed to N₂H₄, it results in some diseases, such as liver injury,⁶ DNA damage,⁷ and even cancer.⁸ Furthermore, US-EPA and IARC defined the low threshold limit value (TLV) of N₂H₄ to be 10 ppb, that is, 0.31 μM.⁹ Therefore, seeking a simple detection method for real-time monitoring of hydrazine, especially in living cells, is important and practically significant for understanding the threat of hydrazine to people's health.

Until now, there have been many efficient methods for the determination of N₂H₄, such as electrochemical method¹⁰ and ultra-high-performance liquid chromatography-tandem mass spectrometry.¹¹ However, these above methods require tedious operation and expensive instruments, and is difficult to achieve real-time detection. On the contrary, fluorescence imaging method is popular due to some merits, including low cost, simple operation, high sensitivity, short detection time, and in situ imaging.^12–15 In the past few years, many fluorescent probes based on different recognizing sites toward N₂H₄ have been reported. The previous probes were mainly divided into the following categories: N₂H₄-triggered cleavage of an acetyl group,^16–18 N₂H₄-triggered cleavage of a 4-bromobutyrate moiety,^19–22 N₂H₄-triggered elimination of an acrylonitrile group,^23–26 N₂H₄-triggered Schiff-base reaction,^27–29 N₂H₄-triggered cyclization reaction,³⁰ N₂H₄-triggered elimination of 2-benzothiazole acetonitrile,^31,32 and others.^33,34 Nevertheless, a few of these probes possessed poor selectivity, and they also detected other ions at the same time. So, developing a novel fluorescent probe with good selectivity for monitoring hydrazine is very interesting.

It is a pity that only few organelle-targeted fluorescent hydrazine-probes have been reported, and they are mainly located in the mitochondria.^35–39 As far as we know, studies on how hydrazine is distributed in living cells are not complete; therefore, developing a novel fluorescent probe, which could monitor the level of hydrazine in organelles would be helpful to deeply understand the biomechanism of hydrazine poisoning to further guide the diagnosis and treatment of hydrazine poisoning. Among all organelles, lysosomes play critical roles in some physical activities, not only digesting most of biomolecules and cellular debris, but also regulating growth factors and repairing plasma membrane.^40,41 Moreover, the redox homeostasis in the lysosomes can be regulated by antioxidants, such as hydrazine from drugs. In other words, developing a lysosomal targeted fluorescent probe for monitoring hydrazine to understand the chemical and biological properties of hydrazine in lysosomes would be significant for guiding the treatment of hydrazine poisoning.

In this study, a novel lysosome-targeted fluorescent probe Cou-Lyso-N₂H₄ with good selectivity for imaging N₂H₄ was designed and constructed (Scheme 2), and the characterization of the probe Cou-Lyso-N₂H₄ is depicted in the ESI† by ¹H NMR, ¹³C NMR and HRMS. As shown in Scheme 1, we used the morpholine unit as an effective lysosome-targeted group due to its alkaline nature, which guided the probe to lysosomes. Furthermore, coumarin was used as a fluorophore, which possesses a high fluorescence quantum yield due to an intramolecular charge transfer (ICT) mechanism, and an ethoxycarbonyl group as the recognition site to N₂H₄, which could be specifically hydrolyzed by hydrazine. This turn-on fluorescent probe Cou-Lyso-N₂H₄ exhibited advantages of good selectivity and low cytotoxicity, and was applied to monitor hydrazine in lysosomes successfully with a high Pearson's coefficient of 0.8674, which might be a useful method to monitor the functions of N₂H₄ in various physiological processes.

Scheme 1 The structure of Cou-Lyso-N₂H₄ and the proposed sensing mechanism for N₂H₄.

Scheme 2 Synthesis of the turn-on fluorescent probe Cou-Lyso-N₂H₄.

The photophysical properties of probe Cou-Lyso-N₂H₄ were studied first. As shown in Fig. S1,† the absorption band of Cou-Lyso-N₂H₄ was at around 335 nm; when the different concentrations of N₂H₄ were added to the reaction system, the initial maximum absorption peak at 335 nm decreased gradually, and a new absorption peak appeared at 410 nm at the same time, which indicated the generation of Cou-Lyso-OH as hydrazine deprotected the acetyl group. In addition, Fig. 1 shows that Cou-Lyso-N₂H₄ had almost no fluorescence at 450 nm in PBS buffer (pH = 7.4) and DMSO (v/v = 4/1) in the absence of N₂H₄, but fluorescence emission at 450 nm (λ_ex = 410 nm) increased significantly with the addition of N₂H₄. Moreover, the fluorescence intensity of probe Cou-Lyso-N₂H₄ at 450 nm showed a good linear relationship with the concentration of N₂H₄ from 20 to 70 μM (Fig. S3†). The corresponding detection limit was calculated to be 3.93 μM with the equation of 3σ/k, where k is the slope and σ is the standard deviation of the blank sample.

Fig. 1 The fluorescence spectra (a) and fluorescence intensity (b) of Cou-Lyso-N₂H₄ (10 μM) in pH 7.4 PBS buffer (containing 20% DMSO) in the absence or presence of N₂H₄ (0–50 equiv.).

Furthermore, we also studied the time-dependent fluorescence response. As illustrated in Fig. 2a and b, the fluorescence intensity at 450 nm exhibited a significant enhancement after the addition of N₂H₄ (10.0 equiv.) within 60 min, then remained almost steady after about 100 min, and the maximum increase was about 9-fold. The result suggested that Cou-Lyso-N₂H₄ could detect hydrazine with high sensitivity.

Fig. 2 The fluorescence spectra (a) and fluorescence intensity (b) of reaction-time profiles of Cou-Lyso-N₂H₄ (10 μM) in the absence or presence of N₂H₄ (10 equiv.) in pH 7.4 PBS buffer (containing 20% DMSO).

In addition, the possible sensing mechanism was studied by means of mass spectrometry. When the probe Cou-Lyso-N₂H₄ (10 μM) was dissolved in PBS buffer (pH = 7.4) and DMSO (v/v = 4/1), an excess of N₂H₄ were added to previous solvent. The spectra showed that there was a clear peak at m/z 319.1295 corresponding to the Cou-Lyso-N₂H₄-adduct, which was Cou-Lyso-OH (Fig. S10†). In addition, the absorption spectra and fluorescence spectra (Fig. S2†) of the dye Cou-Lyso-OH and the probe Cou-Lyso-N₂H₄ before and after adding N₂H₄ were obtained. The result was that the peak of the dye Cou-Lyso-OH was consistent with the newly appeared peak, which was from the probe Cou-Lyso-N₂H₄ after addition of N₂H₄, indicating that the proposed mechanism is correct (Scheme 1).

Another important factor, the pH value of PBS buffer, was investigated, which might have significant impact on the response between Cou-Lyso-N₂H₄ and N₂H₄. As shown in Fig. 3, we measured the fluorescence spectra of probe Cou-Lyso-N₂H₄ (10 μM) in the absence and presence of N₂H₄ (10.0 equiv.) in the range from 3.5 to 11.0. Obviously, the probe Cou-Lyso-N₂H₄ itself was stable, and there was almost no change in a wide pH range from 3.5 to 11.0 in the absence of N₂H₄. However, the fluorescence intensity of probe Cou-Lyso-N₂H₄ increased significantly in pH ranges from 3.5 to 4.5, the fluorescence intensity increased sharply from 4.5 to 7.5, and remained steady from 8.5 to 11.0 upon addition of hydrazine, which indicated that the probe Cou-Lyso-N₂H₄ was suitable for detecting hydrazine in acidic subcellular organelle lysosomes so as to study the harmfulness of N₂H₄ in subcellular organelles during various physiological processes, and the probe Cou-Lyso-N₂H₄ was potential for monitoring hydrazine in a relatively wide pH range especially in a weak alkaline physiological environment.

Fig. 3 The pH effects on the fluorescence spectra of Cou-Lyso-N₂H₄ (10 μM) at different pH values of PBS buffer (containing 20% DMSO) in the absence (■) or presence (●) of N₂H₄ (10 equiv.).

To evaluate whether probe Cou-Lyso-N₂H₄ could effectively work in a complex environment, the selectivity of the probe was investigated. Various analytes including metal ions (Al³⁺, Ca²⁺, K⁺, Mg²⁺, Cu²⁺, Na⁺), anions (Cl⁻, CO₃²⁻, I⁻, SO₃²⁻), nucleophilic biological species (Cys, GSH, Ala), and esterase were measured. As listed in Fig. 4, the fluorescence intensity was basically unchanged with the addition of other test species. On the contrary, with the addition of N₂H₄, there was a significant fluorescence change, which indicated that the probe possessed good selectivity. In addition, we performed the selectivity test together with competitive ions to further evaluate the anti-interference characteristics of the probe Cou-Lyso-N₂H₄. As shown in Fig. 4, when hydrazine and other analytes co-existed, all of the selected species had no interference in the detection of hydrazine, and the fluorescence intensity of the probe still increased significantly, which indicated that the probe possessed a strong anti-interference ability. These results showed that the Cou-Lyso-N₂H₄ probe could be applied to monitor N₂H₄ with good selectivity even in the presence of other species.

Fig. 4 Fluorescence intensity at 450 nm of Cou-Lyso-N₂H₄ (10 μM) in pH 7.4 PBS buffer (containing 20% DMSO) with various relevant species (100 μM) and fluorescence intensity at 450 nm of Cou-Lyso-N₂H₄ (10 μM) in pH 7.4 PBS buffer (containing 20% DMSO) in the presence of N₂H₄ (100 μM) with various relevant species (100 μM). (1) none; (2) Al³⁺; (3) Ala; (4) Ca²⁺; (5) Cl⁻; (6) CO₃²⁻; (7) Cu²⁺; (8) Cys; (9) GSH; (10) I⁻; (11) K⁺; (12) Mg²⁺; (13) Na⁺; (14) SO₃²⁻; (15) esterase.

With the above good properties of probe Cou-Lyso-N₂H₄ in hands, we thought that the probe Cou-Lyso-N₂H₄ could be suitable for detecting N₂H₄ in a living biosystem as it is highly sensitive and selective. Firstly, the MTT assay of Cou-Lyso-N₂H₄ was carried out. As illustrated in Fig. S3,† the HeLa cell survival rate remained more than 85% after treating with Cou-Lyso-N₂H₄ for 24 h even at high concentrations (30.0 μM), which indicated that Cou-Lyso-N₂H₄ possessed low cytotoxicity to living cells, and could be suitable for monitoring N₂H₄ in living cells. Then, the capability of Cou-Lyso-N₂H₄ for imaging N₂H₄ in HeLa cells was investigated. As depicted in Fig. 5, the living HeLa cells were incubated with 10 μM Cou-Lyso-N₂H₄ for 30 min at 37 °C, and washed three times by PBS buffer for removing the excess of probe Cou-Lyso-N₂H₄. As shown in Fig. 5b, the HeLa cells incubated only with Cou-Lyso-N₂H₄ exhibited almost no fluorescence in the green channel. However, after further treating with N₂H₄ (100 μM) for another 30 min, a strong green fluorescence signal emerged obviously (Fig. 5e). Therefore, the probe Cou-Lyso-N₂H₄ exhibited good membrane permeability and possessed ability to image exogenous N₂H₄ in living HeLa cells.

Fig. 5 Imaging of exogenous N₂H₄ in HeLa cells stained with the probe Cou-Lyso-N₂H₄ (10 μM) (a) brightfield image of HeLa cells co-stained only with Cou-Lyso-N₂H₄; (b) fluorescence images of (a) from green channel; (c) overlay of the brightfield image (a) and green channel (b); (d) brightfield image of HeLa cells co-stained with Cou-Lyso-N₂H₄ and treated with N₂H₄; (e) fluorescence images of (d) from blue channel; (f) overlay of the brightfield image (d) and green channel (e).

Finally, we also studied the localization property of the probe Cou-Lyso-N₂H₄ due to the above ideal imaging capability to N₂H₄. The feasibility of Cou-Lyso-N₂H₄ to target lysosomes was investigated in living HeLa cells. As illustrated in Fig. 6, the living HeLa cells were stained with probe Cou-Lyso-N₂H₄ (10 μM) and N₂H₄ for 30 min at 37 °C initially, and were further co-stained with the commercially available Lysosome-Tracker (2.0 mM) for another 30 min. As shown in Fig. 6b and c, the HeLa cells treated with Cou-Lyso-N₂H₄ and N₂H₄ showed a strong green fluorescence signal, and after further incubation with Lysosome-Tracker, the cells exhibited red fluorescence in red channel. Furthermore, the merged images indicated that red fluorescence overlapped well with the green fluorescence. Moreover, the intensity distribution of the linear regions of interest in HeLa cells stained with Lysosome-Tracker and Cou-Lyso-N₂H₄ was almost the same (Fig. 6f). Notably, the Pearson's coefficient was 0.8674 in HeLa cells, suggesting that Cou-Lyso-N₂H₄ was located at the lysosomes. With the above excellent feature of lysosome targeting in hands, this novel probe Cou-Lyso-N₂H₄ possessed a huge potential for imaging N₂H₄ in living HeLa cells.

Fig. 6 Imaging of N₂H₄ in living HeLa cells stained with the probe Cou-Lyso-N₂H₄ (10 μM) and Lysosome-Tracker (2.0 mM) (a) brightfield image of HeLa cells co-stained with Cou-Lyso-N₂H₄; (b) fluorescence images of (a) from green channel; (c) fluorescence images of (a) from red channel; (d) overlay of (a)–(c); (e) intensity correlation plot of probe Cou-Lyso-N₂H₄ and Lysosome-Tracker; (f) intensity profile of regions of interest (ROI) across HeLa cells.

In summary, a lysosomal targeted organic fluorescent probe Cou-Lyso-N₂H₄ for the detection of N₂H₄ was constructed. The ideal probe Cou-Lyso-N₂H₄ exhibited good properties, including good selectivity and low cytotoxicity. In addition, the probe Cou-Lyso-N₂H₄ was used for monitoring N₂H₄ in lysosomes successfully. Hence, the lysosomal targeted probe Cou-Lyso-N₂H₄ might be applied to other functional bio-probes for the recognition of different analytes and to investigate biological and pathological connection of N₂H₄ in living bio-samples.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (21801145), the Shandong Provincial Key R&D Program/Major Science and Technology Innovation Project of Shandong Province (2019JZZY020230), the project ZR2017BB012 supported by Shandong Provincial Natural Science Foundation and the Doctoral Scientific Research Foundation of Shandong Jiaotong University (BS2018019).

Notes and references

1. Y. Ren and H. Yamataka, J. Org. Chem., 2007, 72, 5660–5667.
2. Y. Kao, C. Chong, W. Ng and D. Lim, Occup. Med., 2007, 57, 535–537.
3. N. Nobari, M. Behboudnia and R. Maleki, Appl. Surf. Sci., 2016, 385, 9–17.
4. T. Campbell, V. Foldi and J. Farago, J. Appl. Polym. Sci., 1959, 2, 155–162.
5. H. Ioannidou and P. Koutentis, Tetrahedron, 2009, 65, 7023–7037.
6. T. Leakakos and R. Shank, Toxicol. Appl. Pharmacol., 1994, 126, 295–300.
7. M. Runge-Morris, N. Wu and R. Novak, Toxicol. Appl. Pharmacol., 1994, 125, 123–132.
8. P. Vivekanandan, K. Gobianand, S. Priya, P. Vijayalakshmi and S. Karthikeyan, Drug Chem. Toxicol., 2007, 30, 241–252.
9. K. Nguyen, Y. Hao, W. Chen, Y. Zhang, M. Xu, M. Yang and Y. Liu, Luminescence, 2018, 33, 816–836.
10. S. Sharma, S. Ganeshan, P. Pattnaik, S. Kanungo and K. Chappanda, Mater. Lett., 2020, 262, 127150.
11. J. Oh and H. Shin, J. Chromatogr. A, 2015, 1395, 73–78.
12. F. Cai, B. Hou, S. Zhang, H. Chen, S. Ji, X. Shen and H. Liang, J. Mater. Chem. B, 2019, 7, 2493–2498.
13. J. Hou, B. Wang, S. Wang, Y. Wu, Y. Liao and W. Ren, Dyes Pigm., 2020, 178, 108366.
14. S. Zhang, H. Chen, L. Wang, C. Liu, L. Liu, Y. Sun and X. Shen, J. Mater. Chem. B, 2021, 9, 1089.
15. S. Zhang, H. Chen, L. Wang, X. Qin, B. Jiang, S. Ji, X. Shen and H. Liang, Angew. Chem., Int. Ed., 2021 DOI:10.1002/anie.202107076.
16. C. Liu, K. Liu, M. Tian and W. Lin, Spectrochim. Acta, Part A, 2019, 212, 42–47.
17. S. Guo, Z. Guo, C. Wang, Y. Shen and W. Zhu, Tetrahedron, 2019, 75, 2642–2646.
18. X. Shi, F. Huo, J. Chao, Y. Zhang and C. Yin, New J. Chem., 2019, 43, 10025–10029.
19. H. Lv, H. Sun, S. Wang and F. Kong, Spectrochim. Acta, Part A, 2018, 196, 160–167.
20. Q. Wu, J. Zheng, W. Zhang, J. Wang, W. Liang and F. Stadler, Talanta, 2019, 195, 857–864.
21. M. Zhu, Y. Xu, L. Sang, Z. Zhao, L. Wang, X. Wu, F. Fan, Y. Wang and H. Li, Environ. Pollut., 2020, 256, 113427.
22. K. Wang, Y. Ye, C. Jiang, M. Guo, X. Zhang, A. Jiang, Y. Yang, Q. Jiao and H. Zhu, Dyes Pigm., 2021, 194, 109587.
23. Z. Li, M. Ren, L. Wang and W. Lin, Anal. Methods, 2018, 10, 4016–4019.
24. J. Wu, J. Pan, Z. Ye, L. Zeng and D. Su, Sens. Actuators, B, 2018, 274, 274–284.
25. J. Qu, Z. Zhang, H. Zhang, Z. Weng and J. Wang, Front. Chem., 2021, 8, 1263.
26. S. Mu, H. Gao, C. Li, S. Li, Y. Wang, Y. Zhang, C. Ma, H. Zhang and X. Liu, Talanta, 2021, 221, 121606.
27. W. Xu, W. Liu, T. Zhou, Y. Yang and W. Li, J. Photochem. Photobiol., A, 2018, 356, 610–616.
28. Y. Jung, I. Ju, Y. Choe, Y. Kim, S. Park, Y. Hyun, M. Oh and D. Kim, ACS Sens., 2019, 4, 441–449.
29. X. Li, M. Li, Y. Chen, G. Qiao, Q. Liu, Z. Zhou, W. Liu and Q. Wang, Chem. Eng. J., 2021, 415, 128975.
30. R. Chen, G. Shi, J. Wang, H. Qin, Q. Zhang, S. Chen, Y. Wen, J. Guo, K. Wang and Z. Hu, Spectrochim. Acta, Part A, 2021, 252, 119510.
31. T. Zhang, L. Zhu and W. Lin, Dyes Pigm., 2021, 188, 109177.
32. X. Kong, M. Li, Y. Zhang, Y. Yin and W. Lin, Sens. Actuators, B, 2021, 329, 129232.
33. B. Zhu, X. Wu, J. Rodrigues, X. Hu, R. Sheng and G. Bao, Spectrochim. Acta, Part A, 2021, 246, 118953.
34. N. Rao, Y. Le, D. Li, Y. Zhang, Q. Wang, L. Liu and L. Yan, Chem. Pap., 2021, 1–9.
35. Y. Ran, H. Xu, K. Li, K. Yu, J. Yang and X. Yu, RSC Adv., 2016, 6, 111016–111019.
36. B. Shi, Y. He, P. Zhang, Y. Wang, M. Yu, H. Zhang, L. Wei and Z. Li, Dyes Pigm., 2017, 147, 152–159.
37. Y. Ban, R. Wang, Y. Li, Z. An, M. Yu, C. Fang, L. Wei and Z. Li, New J. Chem., 2018, 42, 2030–2035.
38. X. Kong, B. Dong, C. Wang, N. Zhang, W. Song and W. Lin, J. Photochem. Photobiol., A, 2018, 356, 321–328.
39. B. Wang, R. Yang and W. Zhao, J. Hazard. Mater., 2021, 406, 124598.
40. C. Renfrew and A. Hubbard, J. Biol. Chem., 1991, 266, 21265–21273.
41. M. Marks, P. Roche, E. Donselaar, L. Woodruff, P. Peters and J. Bonifacino, J. Cell Biol., 1995, 131, 351–369.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/d1ay01821c

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