Rational Design of a Hepatoma-Specific Fluorescent Probe for HOCl

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Rational design of a hepatoma-specific fluorescent probe for HOCl and its bioimaging applications in living HepG2 cells Qingxia Duan, Pan Jia, Zihan Zhuang, Caiyun Liu, Xue Zhang, Zuokai Wang, Wenlong Sheng, Zilu Li, Hanchuang Zhu, Baocun Zhu, and Xiaoling Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04726 • Publication Date (Web): 28 Dec 2018 Downloaded from http://pubs.acs.org on January 4, 2019

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Analytical Chemistry

Rational design of a hepatoma-specific fluorescent probe for HOCl and its bioimaging applications in living HepG2 cells Qingxia Duan,† Pan Jia,† Zihan Zhuang,† Caiyun Liu,*,† Xue Zhang,† Zuokai Wang,† Wenlong Sheng,*,‡ Zilu Li,† Hanchuang Zhu,† Baocun Zhu,*,† and Xiaoling Zhang *,§ †

School of Resources and Environment, University of Jinan, Shandong Provincial

Engineering Technology Research Center for Ecological Carbon Sink and Capture Utilization, Jinan 250022, P. R. China. ‡

Qilu University of Technology (Shandong Academy of Sciences), Biology Institute

of Shandong Academy of Sciences, 19 Keyuan Road, Lixia District, Jinan, 250014, Shandong Province, P. R. China. §

Key Laboratory of Cluster Science of the Ministry of Education, Beijing Key

Laboratory of Photoelectronic/Electrophotonic Conversion Materials, School of Chemistry, Beijing Institute of Technology, Beijing 100081, P. R. China. *Corresponding author. Fax: +86-531-82767617; Tel.: +86-531-82767617 E-mail address: [email protected] (B. Zhu), [email protected] (C. Liu), [email protected] (W. Sheng), and [email protected] (X. Zhang)

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ABSTRACT Liver cancer is a kind of high mortality cancer due to the difficulty of early diagnosis. And according to the reports, the concentration of reactive oxygen species (ROS) was higher in cancer cells than normal cells. Therefore, developing an effective fluorescent probe for hepatoma-selective imaging of hypochlorous acid (HOCl) which is one of the vital ROS is of great importance for understanding the role of HOCl in liver cancer pathogenesis. However, the cell-selective fluorescent probe

still

remains

a

difficult

task

among

current

reports.

Herein,

a

galactose-appended naphthalimide (Gal-NPA) with p-aminophenylether as a new receptor and galactose moiety as hepatoma targeting unit was synthesized and employed to detect endogenous HOCl in living HepG2 cells. This probe was proved to possess good water solubility and could respond specifically to HOCl. In addition, probe Gal-NPA could completely react to HOCl within 3 seconds meanwhile accompanied by tremendous fluorescence enhancement. The quantitative linear range between fluorescence intensities and the HOCl concentrations was 0 to 1 µM (detection limit = 0.46 nM). More importantly, fluorescence confocal imaging experiments showed that probe Gal-NPA could discriminate hepatoma cells over other cancer cells and simultaneously traced endogenous HOCl levels in living HepG2 cells. And we thus anticipate that probe Gal-NPA has the potential application for revealing the functions of HOCl in hepatoma cells.


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INTRODUCTION The liver is an important metabolic organ, which plays a part in deoxidization, storage of liver sugars and synthesis of secretory proteins in human body. Recently, various liver diseases gradually threaten human health, for example, drug-induced liver injury,1 alcohol-induced liver disease,2 viral hepatitis.3 And all these diseases have an association with the risk of liver cancer. Liver cancer is one of the high malignant cancers and is also the second most common cause of death from cancers in the world.4,5 More seriously, liver cancer which is called the invisible killer has no special symptoms in premorbid stage and a majority of them are often diagnosed at the late stage, so surgical resection is clearly not feasible.6 Therefore, it is an urgent requirement to develop a visualized and effective method for early diagnosis of liver cancer and specific study of its molecular pathogenesis. According to some literatures, cancer cells can produce more reactive oxygen species (ROS) in comparison to normal cells.7-9 As one of the vital ROS, hypochlorous acid (HOCl) is generated by the assistance of myeloperoxidase (MPO) which can catalyze the oxidation reaction of hydrogen peroxide (H2O2) and chloride ions (Cl-) in living systems.10-12 While the high reactivity of endogenous HOCl makes it to be a double-edged sword, on the one hand, it plays an important role in the immune defense against invasive pathogens.13,14 On the other hand, aberrant accumulation

of

endogenous

HOCl

could

cause

or

exacerbate

various

inflammation-related diseases, such as liver injury and even cancer.15,16 Hence tracing HOCl levels could serve as a pathway for studying the pathogenesis of liver cancer.



But due to the lack of effective tools for tracing endogenous HOCl, the research for its biological functions in the level of specific living cells still remains a challenge.17,18 To date, fluorescent probe has already been confirmed to become an effective tool for detecting biological relevant species based on its high sensitivity and excellent temporal-spatial resolution.19,20 And fluorescence imaging on the cellular level to specific species has already become a dynamic research topic in aspect of chemical biology. In recent years, various fluorescent probes for HOCl imaging in living system have been developed.21-36 For example, Fan and Peng et al developed an “enhanced-PET” based ultrasensitive fluorescent probe for monitoring basal HOCl in cancer cells.14 Additionally, some elegant HOCl probes can specifically localize in lysosomes or mitochondria.37-41 For instance, Yuan et al reported two targetable two-photon fluorescent probes for HOCl imaging in mitochondria and lysosomes.41 Very recently, Yoon et al developed an endoplasmic reticulum-targetable ratiometric fluorescent probe for imaging HOCl in living cells and tissues.42 Unfortunately, organic small molecule fluorescence probes for imaging of endogenous HOCl in the level of specific cells (e.g. hepatoma cells) are still not reported. Thus, it is an urgent need to design novel fluorescent probes with hepatoma-selective imaging of HOCl. However, cell-selective imaging of HOCl still remains a great difficulty due to the effect of complex biological systems.43 Hence the key to develop cell-selective fluorescent probe is that seeking a specific receptors which can enhance selectively internalization of probes within specific cells. Recently, a few of hepatoma-specific fluorescent probes have been developed by introducing a targeting agent of galactose


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moiety for imaging of NO, H2S, thiol and β-galactosidase, etc.44-47 Indeed, the galactose moiety can be specifically recognized in hepatoma cells which have over-expressed asialoglycoprotein receptor (ASGP-R) and the achievement of cell-selective imaging relies on the use of a specific receptor-mediated endocytosis.48 Herein, we report a new galactose-based hepatoma-specific fluorescent probe (Gal-NPA) employing p-aminophenylether as a recognition receptor for tracing endogenous HOCl in living HepG2 cells. The 4-position of galactose-appended 1,8-naphthalimide was displaced by p-aminophenylether group and its fluorescence was effectively quenched based on the enhanced photo-induced electron transfer (PET) effect and inhibited intramolecular charge transfer (ICT) process. As expected, probe Gal-NPA exhibited excellent water solubility owing to the introduction of galactose moiety and a dramatic fluorescence enhance in the presence of HOCl with high selectivity and sensitivity (DL = 0.46 nM). More importantly, experimental results of confocal microscopic imaging revealed that probe Gal-NPA could perform hepatoma-selective

image

of

HOCl

through

galactoside

receptor-promoted

endocytosis. As a control, a lack of galactose targeting agent probe NPA was synthesized and studied to further proof the hepatoma-specificity of probe Gal-NPA.

EXPERIMENTAL SECTION The detailed synthetic process of probe Gal-NPA was shown in Scheme 1. Compound 1, 2, a and probe NPA were prepared according to the literature and synthetic routes were described in Scheme S1 and Scheme S2.



Scheme 1. Synthesis of probe Gal-NPA and the structure of NPA Synthesis of probe Gal-NPA. The mixture of compound 2 (453 mg, 1.32 mmol) and a (330 mg, 0.88 mmol) in CH2Cl2/H2O (10 mL, v/v = 1:1) were added sodium ascorbate (398 mg, 2.01 mmol) and CuSO4·5H2O (166 mg, 0.67 mmol). Then reaction solution was stirred at room temperature overnight. After that the mixture was diluted with CH2Cl2 and extracted with water. The combined organic layer was dried used anhydrous NaSO4, and the resulting solution was evaporated, then purified by column chromatography to give 3 (440 mg) in 70% yield. Finally, compound 3 was dissolved in MeOH/H2O (8:1, v/v), followed by addition of excessive Et3N and stirred at room temperature for 1 h. Then the solvent was removed and the resulting crude product was purified by column chromatography (CH2Cl2/MeOH = 7:1, v/v) to give pure probe Gal-NPA (102 mg) in 31% yield. 1H NMR (400 MHz, DMSO-d6) δ (*10-6): 3.411-3.518(m, 3H), 3.659(t, J = 6.0 Hz, 1H), 3.705(t, J = 4.4 Hz, 1H), 3.921-3.982(m, 1H), 4.572(d, J = 5.6 Hz, 1H),


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4.695(t, J = 5.6 Hz, 1H), 4.965(d, J = 5.6 Hz, 1H), 5.178(d, J = 5.6 Hz, 1H), 5.218(s, 1H), 5.313(s, 1H), 5.426(d, J = 9.2 Hz, 1H), 6.695 (d, J = 8.8 Hz, 1H), 6.890 (d, J = 8.4 Hz, 1H), 6.975 (d, J = 8.8 Hz, 1H), 7.913(t, J = 8.0 Hz, 1H), 8.147(s, 1H), 8.420(d, J = 8.4 Hz, 1H), 8.590(d, J = 7.2 Hz, 1H), 8.746(d, J = 7.6 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ (*10-6): 60.87, 68.94, 69.72, 74.12, 78.91, 88.54, 115.46, 122.10, 122.31, 122.49, 127.32, 129.06, 129.56, 132.11, 133.74, 143.75, 144.12, 147.36, 161.41, 163.17, 163.89. HRMS (ESI): Calcd for C27H26N5O8 [M+H]+ 548.1776; Found, 548.1773.

RESULTS AND DISCUSSION Design and synthesis of probe Gal-NPA for HOCl. In the molecular designing, we employed 4-hydroxy-1,8-naphthalimide as fluorophore to construct probe Gal-NPA due to its effective intramolecular charge transfer (ICT) structure.49 The high quantum yield and good photo-stability also facilitate the application of 4-hydroxy-1,8-naphthalimide.50-52 So it is widely used for designing new fluorescent probes and some excellent probes were synthesized based on the combination of naphthalimide and other fluorophore such as rhodamine.53-55 On the other hand, it is crucial to seek a specific recognition receptor for constructing a novel HOCl fluorescent probe. According to the reports, p-aminophenylether group with electron-rich could recognize highly reactive oxygen species such as •OH, ONOO-, and HOCl.56-59 Furthermore, Prof. Wang et al. demonstrated that the oxidative cleavage reaction is an ideal strategy for constructing highly effective fluorescent probes.60-62 Hence, we prepared a 4-hydroxy-1,8-naphthalimide-derived



p-aminophenylether-based fluorescent probe Gal-NPA for tracing endogenous HOCl in specific hepatoma cells through introducing a targeting agent of galactose moiety (Scheme 1). The properties of fluorescent probe Gal-NPA for HOCl. The spectral properties of Gal-NPA were studied in aqueous solution containing 5 mM PBS (pH 7.4). Firstly, the photobleaching experiment of this probe was performed by continuous excitation for 1 h and the result showed that probe Gal-NPA displayed excellent photostability (Figure S1). Then response time of Gal-NPA in the presence of HOCl was evaluated because it is a meaningful factor for reaction-based probes. As shown in Figure 1, the probe solution of Gal-NPA exhibited negligible fluorescence (Φ = 0.014) without the addition of HOCl at 558 nm, and this actually attributed to the effective inhibition of ICT structure and enhanced PET effect to the fluorophore resulting from p-aminophenylether group. While the fluorescence intensity has been enhanced significantly about 50-fold (Φ = 0.143) after adding 5 μM HOCl and the reaction was finished instantaneously within 3 seconds. The result suggested that probe Gal-NPA has tremendous potential for real-time detection of HOCl in situ. The absorption spectra of probe Gal-NPA was investigated upon addition of 10 μM HOCl. As shown in Figure 2, a major peak of Gal-NPA was obtained at 375 nm in absence of HOCl and a new clear absorption peak at around 455 nm appeared with the addition of HOCl. Meanwhile, the reaction also accompanied by significant solution color change that could be changed from colorless to yellow. These phenomena such as considerable fluorescence enhancement,


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red-shifted absorption peak, as well as solution color change, indicated the production of stronger ICT structure. And these features make it convenient to achieve rapid monitoring of HOCl in the biosystems.

Figure 1. Time-course of probe Gal-NPA (5 µM) in the presence of HOCl (5 µM).

Figure 2. Absorption spectra of probe Gal-NPA (20 µM) in the presence HOCl (10 µM). The illustration shows the color change of probe solution upon addition of HOCl. To evaluate the ability of probe Gal-NPA for tracing low concentrations of HOCl, the quantitative experiment was performed in 0-5 µM concentrations of HOCl. As shown in Figure 3, the solution of probe Gal-NPA has no obvious fluorescence at 558 nm, while the fluorescence intensity exhibited remarkable enhancement with the



addition of increasing HOCl concentrations. Moreover, a satisfactory linearity between fluorescence intensities at 558 nm and HOCl concentrations (0-1 µM) was acquired and the linear equation was y = 288804 × [HOCl] (μM) + 30470 (R2 = 0.9928). The detection limit (DL) of 0.46 nM was also obtained by calculating with the equation DL = 3σ/k. Obviously, probe Gal-NPA possesses higher sensitivity for monitoring HOCl level compared with the most of reported fluorescent probes (Table S1).

Figure 3. (a) Fluorescence spectra of probe Gal-NPA (5 μM) in the presence of increasing concentrations of HOCl (0-5 µM) under aqueous solution containing PBS (5 mM, pH 7.4). (b) The linearity between fluorescence intensities at 558 nm and the increasing concentrations of HOCl (0-1 µM). Excitation wavelength = 470 nm. Each


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spectrum was acquired 1 min after HOCl addition. The excellent selectivity is a crucial property for probe Gal-NPA to the effective detection of HOCl. Therefore, we investigated fluorescence responses of probe Gal-NPA towards different analytes, including other ROS or RNS species, amino acids, biothiols, essential metal ions and anion. As shown in Figure 4, only hypochlorous acid induced a dramatic fluorescence enhancement at 558 nm and negligible variations of fluorescence signals were obtained in the presence of other higher concentration analytes by contrast. Although p-aminophenylether has been developed as the recognition receptor of ROS including •OH, ONOO-, and HOCl, the most likely reason is that probe structure determines its selectivity. The obtained results completely manifested probe Gal-NPA could sever as a highly selective fluorescent probe for detecting HOCl without interference from other bioactive molecules.

Figure 4. Fluorescence responses of probe Gal-NPA (5 μM) in the presence of various analytes (100 μM except for specific labels) in the PBS solution (5 mM, pH 7.4) 1. Blank, 2. K+, 3. Na+, 4. Ca2+, 5. Mg2+, 6. Zn2+, 7.Cu2+, 8. Fe3+, 9. Fe2+, 10. Cl-, 11. NO3-, 12. NO2-, 13. SO42-, 14. SO32-, 15. HSO3-, 16. S2-, 17. Cys (500 μM), 18.



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Hcy (500 μM), 19. GSH (5 mM), 20. H2O2, 21. TBHP, 22. •OH, 23. •OtBu, 24. O2-, 25. 1

O2, 26. NO, 27. ONOO− (5 μM), 28. HOCl (5 μM). To disclose the response mechanism of Gal-NPA for HOCl, the reaction of

Gal-NPA and HOCl was performed under the same conditions as described above. The resulted mixtures were subjected to electrospray ionization mass spectral analysis. The mass peak at m/z 455.1205 corresponding to 4-hydroxy-1,8-naphthalimide derivate ([M-H]- calcd 455.1208) was obtained. According to above experimental results

and

previous

literatures

about

the

reaction

mechanism

of

p-aminophenylether-based fluorescent probe for recognizing HOCl, a reasonable sensing mechanism was proposed and demonstrated (Scheme 2). Additionally, in order to further demonstrate the sensing mechanism, we synthesized the compound 4 which replaced p-aminophenylether with phenylate group as a control (Scheme S3). Then we respectively investigated the response of probe NPA and compound 4 towards HOCl (Figure S2 and S3). Combined with the above experimental results, it is obvious that both probe Gal-NPA and NPA could recognize HOCl while the fluorescence intensity of compound 4 has no changes with the addition of different concentration HOCl. Therefore, the most probable reaction mechanism is that HOCl-mediated oxidative removal effect cleaves p-aminophenylether moiety and enhances the ICT process of product structure.


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Scheme 2. The sensing mechanism of probe Gal-NPA for HOCl The hepatoma-specific ability of probe Gal-NPA. According to above experimental results that probe Gal-NPA could rapidly, selectively and ultrasensitively detect HOCl in aqueous solution, and then we attempt to apply probe Gal-NPA to the imaging of HOCl in living HepG2 cells because these cells have over-expressed ASGP-R which can specifically recognize galactosides.43 But before that, the cytotoxicity of probe Gal-NPA was investigated primarily. Cytotoxicity of probe Gal-NPA in HepG2 cells were obtained by a cell counting kit-8 (CCK-8). And the cell viability was not obviously affected after adding probe Gal-NPA (Figure S4), demonstrating that probe Gal-NPA possesses the feature of low cytotoxicity and good biocompatibility. The hepatoma-specific ability of probe Gal-NPA was investigated through respectively incubating human hepatoma cells (HepG2) and asialoglycoprotein receptor-negative cells including human gastric carcinoma cells (MGC803), lung cancer cells (A549) and marrow neuroblastoma cells (SHSY5Y) with probe Gal-NPA for 30 min. As shown in Figure 5, the significant fluorescence signal was observed in HepG2 cells incubating with probe Gal-NPA, while other kinds of cells exhibited no



fluorescence intensity change under the same experimental conditions. As a control, probe NPA without hepatoma-targeting ligand was respectively incubated in these different kinds of cells. The confocal fluorescence imaging showed that probe NPA could enter the cell and cause the fluorescence enhance regardless of cell types (Figure 5). All the above experimental results demonstrated that probe Gal-NPA has hepatoma-selective ability between hepatoma cells and other cancer cells, and this probably relies that the presence of galactose-based ligand promotes selective internalization of probe Gal-NPA via ASGPR-mediated endocytosis.

Figure 5. Cell fluorescence imaging of probe Gal-NPA (20 μM) and NPA (20 μM) in HepG2, MGC803, A549 and SHSY5Y cells. Fluorescent probe Gal-NPA for imaging endogenous HOCl in hepatocytes. Inspired by the excellent hepatoma-selective ability of Gal-NPA, the fluorescence imaging of Gal-NPA for HOCl was investigated specifically in living HepG2 cells. As shown in Figure 6, the control cells exhibited medium fluorescence


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signal and the cells pretreated with N-acetyl-L-cysteine (NAC, a scavenger of HOCl) showed weaker intracellular fluorescence. At the same time, cells pretreated with phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS) followed by incubating Gal-NPA displayed obviously stronger green fluorescence than control cells. The results proved that probe Gal-NPA could trace the endogenous HOCl levels in hepatoma cells. Additionally, we observed significantly enhanced fluorescence in cells incubated with Gal-NPA followed by adding HOCl. All these experimental results suggested that probe Gal-NPA with hepatoma-selective ability could trace endogenous or exogenous HOCl levels utilizing the visualized fluorescence imaging method.

Figure 6. Fluorescence imaging in HepG2 cells: incubated with 20 μM Gal-NPA (the control) for 30 min; respectively pretreated with 500 μM NAC and 1 μg/mL PMA+LPS for 1 h followed by incubating 20 μM Gal-NPA for 30 min; incubated with 20 μM Gal-NPA for 30 min followed by the addition of 20 μM HOCl for 20 min.

CONCLUSION In conclusion, a new hepatoma-selective p-aminophenylether-based fluorescent probe Gal-NPA was successfully constructed for the determination of HOCl in



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biological systems. Probe Gal-NPA displayed excellent water solubility and low cytotoxicity, and could transiently response to HOCl that make it possible for hepatoma-selective fluorescence imaging. Additionally, prominent selectivity and sensitivity of Gal-NPA greatly facilitate the detection of HOCl in hepatoma cells. Inspired by fluorescence imaging experiments, we expect that probe Gal-NPA would be a useful tool for revealing the functions of HOCl in hepatoma cells and studying the HOCl-related liver diseases. AUTHOR INFORMATION Corresponding Author *

[email protected]

(B.

Zhu),

[email protected]

(C.

Liu),

[email protected] (W. Sheng), and [email protected] (X. Zhang) Author Contributions All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interests. ACKNOWLEDGMENT We gratefully acknowledge financial support from the National Natural Science Foundation of China (21777053 and 21607053), Shandong Provincial Natural Science Foundation

(ZR2017MB014),

Shandong

Provincial

Key

Research

Project

(2016GSF117010), and A Project of Shandong Province Higher Educational Science and Technology Program (J16LD01). ASSOCIATED CONTENT


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Supporting Information. The preparation of compound 1, 2, 4 and a, experimental details of cell culture and cytotoxicity assays. This material is available free of charge via the Internet at http://pubs.acs.org.

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For TOC only


Rational Design of a Hepatoma-Specific Fluorescent Probe for HOCl

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