A dual-channel fluorescence probe for simultaneously visualizing cysteine and viscosity during drug-induced hepatotoxicity

Abstract

Cysteine (Cys), one of the important participants in protecting cells from oxidative stress, is closely associated with the occurrence and development of various diseases. Moreover, cell viscosity as a pivotal microenvironmental parameter has recently attracted increasing attention due to its dominant role in governing intracellular signal transduction and diffusion of reactive metabolites. Thus, simultaneous detection of Cys and viscosity is imperative for investigating their pathophysiological functions and cross-link. Herein we present a mitochondria-targetable dual-channel fluorescence probe ABDSP by grafting the acrylate modified pyridinium unit to dimethylaminobenzene. Whilst the probe is a seemingly simple, it could simultaneously discriminate Cys and viscosity in a fashion of distinguishable signals. Furthermore, the probe was successfully employed for visualizing mitochondrial Cys and viscosity, and probe into their cross-link during acetaminophen-induced hepatotoxicity.

Graphical abstract

Highlights

• •Imaging the cysteine and viscosity in green and red channels, respectively.
• •Superior sensitivity, large Stokes shift and excellent selectivity toward cysteine.
• •136-fold red fluorescence enhancement toward viscosity.
• •Visualizing the cross-link between cysteine and viscosity during hepatotoxicity.

1. Introduction

Biothiols, mainly including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), stand out as the second messengers of oxidative stress, and function as important players in redox homeostasis, signal transduction and metabolic regulation in biological system [[1], [2], [3]]. They harbor similar chemical properties owing to their similar chemical structures, but play different roles in some physiological events. Apart from being the biosynthesis precursors of GSH and sulfur-containing proteins [4], Cys also participates in maintaining the biological conformation of protein, and mediates the body's intestinal function, lipid metabolism and other processes [[5], [6], [7]]. It is worth noting that Cys is tightly related to the occurrence and development of various diseases. Excessive Cys could induce Alzheimer's disease, Rheumatoid arthritis and Parkinson's disease, while deficient Cys will result in retarded growth, liver damage, edema and skin damage [[8], [9], [10]].

On one hand, the growth, proliferation and normal physiological function of cells are inseparable from the balance of redox homeostasis [11,12]; on the other hand, the multiple biological activities of cells are closely related to the microenvironments [[13], [14], [15]]. Cellular microenvironmental factors mainly include polarity, viscosity and pH, where viscosity acts as a key biomarker to engage in signal transduction, electron transport and biomolecule interaction [[15], [16], [17], [18], [19]]. The abnormal variation of cell viscosity may lead to the occurrence of multitudinous diseases including malignant tumor, diabetes, Alzheimer's disease and atherosclerosis [15,[20], [21], [22]]. Additionally, there is a close correlation between intracellular viscosity and redox homeostasis, such as an increase viscosity induced by oxidative stress. Thus, considering the vital cross-link between redox signaling molecules and cellular microenvironment parameters in biological system, it is of great significance to develop a powerful tool for simultaneously detecting them.

Nowadays, fluorescence imaging has attracted a great deal of attention owing to the superior performance of simple operation, nondestructive detection, high temporal-spatial resolution and excellent sensitivity [[23], [24], [25], [26], [27], [28]]. However, only a few fluorescence probes were employed for simultaneously monitoring Cys and viscosity [[29], [30], [31], [32]], and it is still imperative to develop a single probe to differentiate them simultaneously by the distinguishable signals. Accordingly, we constructed a dual-channel fluorescence probe ABDSP to simultaneously detect Cys and viscosity, and visualize their interconnectedness in the pathological process. The probe features the 4-p-dimethylaminostyrylpyridinium and acrylate, which serve as viscosity response unit and Cys recognition site, respectively (Scheme 1A). We reasoned that probe ABDSP could exhibit green fluorescence with Cys owing to the DPA generation through the addition-cyclization reaction of the probe and Cys [33], and red fluorescence with viscosity by the suppression of the twisted intramolecular charge transfer (TICT) process [34,35]. Indeed, the sulfhydryl group of Cys can attack the probe ABDSP to form a thioether, and then amino group undergo intramolecular cyclization to generate the cyclized thiolactam product, which is accompanied by a subsequent 1,6-elimination reaction of the p-hydroxybenzyl moiety to release the free fluorophore DPA (Scheme 1B). Meanwhile, viscosity could induce a turn-on fluorescence enhancement of the probe ABDSP at 613 nm due to the suppression of the single-bond rotation between dimethylaminobenzene and vinylpyridinium.

Scheme 1.

Rational design of a dual-channel fluorescence probe ABDSP. (A) Molecular structure of ABDSP and its mechanism for simultaneously detecting Cys and viscosity. (B) The detailed mechanisms of probe ABDSP response to Cys.

2. Materials and methods

2.1. Synthesis of ABDSP

The probe ABDSP was easily prepared by 4-step reactions following the synthetic routes shown in Scheme S1, and its synthesis details and characterization were included in the Supporting Information.

2.2. Optical studies

The probe ABDSP was dissolved in DMSO for preparing the stock solution (10 mM), and then diluted to 10 μM in optical response experiments. Biothiols and anions were dissolved in deionized water to prepare the stock solution with a concentration of 100 mM, and metal ions were dissolved in deionized water for preparing the stock solution (50 mM). Subsequently, the analytes were diluted to the desired concentration with PBS for the optical response experiments. Moreover, the viscosity response experiments were carried out in the mixed solvent of PBS and glycerol. The UV/vis absorption and fluorescence response spectra were recorded using the TU-1901 and F-7000 FL spectrophotometer, respectively. The excitation and emission slit width were set at 10 nm and 20 nm, respectively.

2.3. Cytotoxicity of probeABDSP

The cytotoxicity of probe ABDSP was evaluated by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HepG2 cells purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China), were cultured in RPMI 1640 medium including 10 % fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin, and grown at 37 °C in a humidified 5 % CO₂ incubator. Subsequently, the cells (5 × 10³ cells/well) harvested from culture flasks were seeded into 96-well plates and adhered for overnight, followed by treatment with different concentrations of probe ABDSP for 24 h. Removing the culture medium, the cells were incubated with RPMI 1640 medium containing 0.5 mg/mL MTT for another 4 h. The OD values were acquired by Tecan Infinite M200 microplate reader.

2.4. Cell imaging

HepG2 cells were seeded into 6-well culture plates at a density of 2 × 10⁵ cells/well and allowed to resume exponential growth for overnight. For imaging of Cys, the cells were treated with different concentrations of Cys for 30 min, and further incubated with the probe ABDSP for 30 min. To image viscosity, HepG2 cells were sequentially treated with nystatin (30 min) and probe ABDSP (30 min). To image the acetaminophen (APAP)-induced hepatotoxicity, HepG2 cells were treated with APAP for 12 h, followed by incubation with probe ABDSP for 30 min, and NAC pretreatment for 1 h if necessary. After treatment in various ways, the cells were washed with PBS and imaged by fluorescence microscope (Leica DMI 4000B, Germany).

3. Results and discussion

3.1. Optical response to Cys

With ABDSP in hand, we initially evaluated the responsive ability of probe toward Cys using a UV/vis absorption spectrometer. As shown in Fig. S1, the probe displayed an obvious absorption peak at 488 nm. Upon addition of Cys, the original UV/Vis absorption peak decreased significantly as well as the emergence of a new absorption peak at about 380 nm. Meanwhile, the probe alone showed a negligible fluorescence emission upon excitation at 380 nm but manifested a fluorescence enhancement at 530 nm in the presence of Cys with a large Stokes shift (150 nm) (Fig. 1A). These changes of the probe in absorption and fluorescence spectra were assigned to the release of the corresponding fluorophore DPA via Cys-mediated addition-cyclization reaction. Subsequently, a good linear relationship between probe fluorescence intensity at 530 nm and Cys concentration was revealed, and the detection limit was calculated to be 8.8 nM based on 3σ/k method, highlighting the superior sensitivity of probe ABDSP to Cys (Fig. 1B). The reaction-time experiment was also carried out, and the fluorescence signals gradually increased and reached the steady state within 25 min after adding 200 μM Cys (Fig. 1C). To investigate the effect of pH value on the probe, we conducted the pH titration experiments. As shown in Fig. S2, the probe itself was stable in a pH range of 4.0–10.0, but exhibited a strong fluorescence response to Cys under physiological conditions, supporting the biological application of probe ABDSP. Additionally, we further evaluated the selectivity of probe ABDSP toward Cys in the presence of various relevant interfering substances including biothiols, metal ions and anions. As shown in Fig. 1D, Cys caused a remarkable enhancement of fluorescence intensity at 530 nm in comparison with other test species, stressing the excellent specificity of the probe to Cys.

Fig. 1.

Optical response of ABDSP toward Cys. (A) Fluorescence emission spectra of ABDSP (10 μM) upon addition of Cys (0–200 μM) in PBS/DMSO buffer (1/1, v/v). (B) Linear relationship between the intensity at 530 nm and Cys concentrations. (C) Changes in fluorescence intensity of probe ABDSP (10 μM) at 530 nm over time in the presence of Cys (200 μM). (D) Fluorescence response of ABDSP (10 μM) at 530 nm toward GSH (1 mM), Cys (200 μM) and other analytes (100 μM) in PBS/DMSO buffer (1/1, v/v). λ_ex = 380 nm, slit (nm): 10/20.

Next, the reaction mechanism was further explored using absorption spectrum, fluorescence spectrum and mass spectroscopy. As shown in Fig. S3, the mixture of probe ABDSP response to Cys exhibited the strong absorption at 380 nm and fluorescence emission at 530 nm, which was consistent with the characteristic absorption and fluorescence emission of the fluorophore DPA (Figs. S3A and B). Moreover, the reaction products of probe ABDSP with Cys were also analyzed by mass spectrometry, and the main molecular ion peak (m/z = 225.1979, [M + H]⁺) corresponding to the fluorophore DPA was observed (Fig. S3C). Meanwhile, we also detected the mass peak of the intermediate product, such as m/z = 506.3046 and 331.2535 assigned to the Cys-adduct (Cys-ABDSP) and phenol intermediate (PDPA), respectively. According to these data, we could reason that the probe first undergoes an addition reaction with Cys to produce the adduct (Cys-ABDSP), followed by the cyclization reaction to release the phenol intermediate (PDPA) and the elimination reaction to obtain the fluorophore DPA. These results are in line with the proposed mechanism shown in Scheme 1.

3.2. Optical response to viscosity

Firstly, the UV/Vis absorption changes of the probe in viscous and non-viscous medium were investigated. It can be seen from Fig. S4 that the absorption peak of probe ABDSP displayed a slight red shift from 488 to 498 nm in viscous glycerol system compared to PBS/DMSO buffer, which was attributed to the restriction of molecular rotation. Secondly, the fluorescence property of the probe was measured in different viscosity. As shown in Fig. 2A, the probe exhibited the faint background fluorescence in PBS, while the fluorescence signal of probe ABDSP gradually enhanced as the rising glycerol ratio and reached its maximum value in pure glycerol. Notably, the fluorescence emission of the probe at 613 nm enhanced 136-fold. Finally, probe ABDSP exhibited negligible fluorescence response in non-viscous environment (Fig. 2B). The above data demonstrate that probe ABDSP could serve as a potential tool for monitoring viscosity.

Fig. 2.

(A) Fluorescence spectra of ABDSP (10 μM) in different ratios of PBS (P) and glycerol (G) mixtures. (B) Fluorescence spectra of ABDSP (10 μM) in different solvents. λ_ex = 498 nm, slit (nm): 10/20.

3.3. Cytotoxicity and colocalization experiment

Prior to colocalization experiment, the cytotoxicity of probe ABDSP against HepG2 cells was tested through the standard MTT assay, and the results showed that the probe possessed low cytotoxicity at the concentration up to 20 μM (Fig. S5). Accordingly, we selected the probe with a concentration of 5 μM for subsequent colocalization experiment and cell imaging. As shown in Fig. 3, the fluorescence of Mito-Tracker Green (a commercialized mitochondrial dye) in green channel overlapped well with that of probe ABDSP in red channel (Fig. 3A–C), and Pearson's colocalization coefficient was determined as 0.87 (Fig. 3D), hinting the excellent mitochondria-targeting capacity of the probe.

Fig. 3.

Intracellular localization of ABDSP. (A–C) Fluorescence images of HepG2 cells treated with ABDSP (5 μM, 30 min) and subsequently stained with Mito-Tracker Green (50 nM, 30 min). Green channel: λ_ex = 460–500 nm, λ_em = 512–542 nm. Red channel: λ_ex = 515–560 nm, λ_em = 590–630 nm. (D) Scatter plot: the correlation plots of green and red channels.

3.4. Cell imaging

With the aid of probe ABDSP, the fluorescence experiments for imaging intracellular Cys were then conducted. As shown in Fig. 4A, cells only treated with probe ABDSP presented the weak green fluorescence, indicating that the probe is capable of tracking mitochondrial basal Cys. Upon addition of Cys, cells exhibited the significantly enhanced fluorescence signals in the green channel along with a dose-dependent fashion, implying that the probe can monitor the fluctuation of mitochondrial Cys levels. In addition, nystatin could disrupt the ionic balance of cells, cause mitochondrial dysfunction, and increase viscosity [36,37]. As shown in Fig. 4B, compared to the cells directly incubated with the probe, that pretreated with nystatin and then stained ABDSP displayed a strong red fluorescence, and the fluorescence intensity increased with the increasing dosages of nystatin. When the cells were treated with nystatin at the same concentration for different time, the gradually enhanced red fluorescence signal was observed over time (Fig. S6), demonstrating a time-dependent relationship between fluorescence intensity of probe ABDSP and nystatin. Furthermore, the photostability of probe ABDSP in living cells has also been evaluated by treating cells with Cys and nystatin, respectively. Fig. S7 showed that the green and red fluorescence intensity remain unchanged within 10 min, highlighting that probe ABDSP has a good photostability. Collectively, the above results support that probe ABDSP can simultaneously detect Cys and viscosity in a well-separated dual-channel mode.

Fig. 4.

Cellular imaging of Cys and viscosity. (A) (a1, a2) HepG2 cells were treated only with ABDSP (5 μM) for 30 min (b1, b2, c1 and c2) HepG2 cells were treated with Cys (100 or 200 μM) for 30 min and then incubated with ABDSP (5 μM) for another 30 min. Scale bar: 25 μm. λ_ex = 460–500 nm, λ_em = 512–542 nm. (B) (a1, a2) HepG2 cells were only incubated with ABDSP (5 μM) for 30 min (b1, b2, c1 and c2) HepG2 cells were treated with nystatin (5 or 10 μM) for 30 min and then incubated with ABDSP (5 μM) for another 30 min. Scale bar: 25 μm. λ_ex = 515–560 nm, λ_em = 590–630 nm.

3.5. Drug-induced hepatotoxicity

APAP is a commonly used pain killer and antipyretic drug [38,39], and can induce hepatotoxicity by an extensive oxidative stress, leading to the increase of viscosity and the irreversible depletion of Cys [39,40]. To visualize the changes of viscosity and Cys in the process of hepatotoxicity, we used APAP as an inducer for the following fluorescence imaging experiments. As shown in Fig. 5, there was a bright green fluorescence and weak red fluorescence in the cells exclusively incubated with probe ABDSP (Fig. 5a1-a3). After treating with APAP and then staining with probe ABDSP, the cells showed the obvious decrease of green fluorescence signal and remarkable enhancement of red fluorescence signal (Fig. 5b1-b3), whereas the pretreatment with NAC (a classic antioxidant) reversed clearly the fluorescence changes induced by APAP (Fig. 5c1-c3). These data visualized the internal relationship between Cys and viscosity during APAP-induced hepatotoxicity, that is, oxidative stress led to the decrease of Cys level and increase of viscosity, illustrating that probe ABDSP can be beneficial for imaging the toxicity mechanism of APAP.

Fig. 5.

Fluorescence images of APAP-induced hepatotoxicity. (a1-a3) HepG2 cells were directly incubated with ABDSP (5 μM, 30 min) as control. (b1-b3) HepG2 cells were stimulated with APAP (500 μM, 12 h) and incubated with ABDSP (5 μM, 30 min). (c1-c3) HepG2 cells were successively incubated with NAC (1 mM, 1 h), APAP (500 μM, 12 h) and ABDSP (5 μM, 30 min). Scale bar: 25 μm. Green channel: λ_ex = 460–500 nm, λ_em = 512–542 nm. Red channel: λ_ex = 515–560 nm, λ_em = 590–630 nm.

4. Conclusions

In summary, we designed and synthesized a dual-functional fluorescence probe ABDSP for simultaneously detecting Cys and viscosity by virtue of the distinct signals: green for Cys and red for viscosity. Probe ABDSP, with superior sensitivity (detection limit = 8.8 nM), a large Stokes shift (150 nm) and excellent selectivity toward Cys, and 136-fold red fluorescence enhancement toward viscosity, was successfully employed to visualize mitochondrial Cys and viscosity in living cells. More importantly, the probe was first applied to probe into the mutual relevance between Cys and viscosity during APAP-induced hepatotoxicity. We expect that the probe would be a promising tool for further understanding the roles of Cys and viscosity under different pathophysiological conditions.

Data availability statement

Data will be made available on request.

CRediT authorship contribution statement

Ya-Long Zheng: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Investigation, Data curation, Conceptualization. Ruixue Yu: Visualization, Project administration, Investigation, Data curation. Mengbo Li: Project administration, Investigation, Data curation. Cailian Fan: Validation, Supervision, Investigation, Data curation. Li Liu: Validation, Supervision, Investigation. Huijie Zhang: Investigation, Formal analysis, Data curation. Wenqian Kang: Supervision, Investigation, Formal analysis, Data curation. Run Shi: Formal analysis, Data curation. Changzhi Li: Investigation, Data curation. Yarui Li: Validation, Data curation. Jiaqi Wang: Validation, Data curation. Xinhua Zheng: Writing – original draft, Visualization, Supervision, Project administration, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article.