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DOI: 10.1039/D4AN00906A (Paper) Analyst, 2024, Advance Article

A single-molecule fluorescent probe for visualizing viscosity and hypoxia in lysosomes and zebrafish embryos†

Jingchao Wang‡ a, Lina Zhou‡a, Zekun Jianga, Haiyan Wu*b and Xiuqi Kong*a
aSchool of Chemistry and Chemical Engineering, University of Jinan, Jinan, Shandong 250022, P. R. China. E-mail: ifp_kongxq@ujn.edu.cn
bDepartment of Pharmacy, Central Hospital Affiliated to Shandong First Medical University, Shandong, Jinan 250013, China. E-mail: wuhaiyanangel@163.com

Received 27th June 2024 , Accepted 7th August 2024

First published on 10th August 2024

Abstract

Viscosity and hypoxia, as microenvironment parameters, play important roles in maintaining normal biological processes and homeostasis. Therefore, simultaneous and sensitive detection of these elements with simple and effective methods could offer precise information in biology. Here, we report a two-site lysosome-targeting fluorescent probe, NVP, for monitoring viscosity and nitroreductase with dual emission channels (emission shift is 86 nm). The NVP probe has displayed highly sensitive and selective responses towards viscosity and nitroreductase, respectively. Significantly, the fluctuations of viscosity and NTR have been detected in vitro and in vivo. We expect that the dual-responsive fluorescent NVP probe will become a potential molecular tool for the exploration of deeper functions of viscosity and hypoxia.

1. Introduction

Hypoxia as one of vital microenvironment-related parameters, plays important roles in maintaining normal biological processes and homeostasis by controlling diffusion, transport and intermolecular interactions.1–3 Low oxygen concentrations are associated with many common diseases including vascular diseases4,5 and cardiopathy,6,7 also some cancers.8–10 Hypoxia also accelerates the production of cancer cells, developing and increasing drug resistance.11–13 Significantly, nitroreductase (NTR) is found to be overexpressed in hypoxia and cancer cells, and considered a biomarker of hypoxia level. NTR is one of the flavoprotein enzymes, which can catalyze the reduction of nitroaromatic compounds to amines combined with nicotinamide adenine dinucleotide (NADH).14–16 Thus, using a powerful molecular tool for real-time detection of hypoxia conditions in biology is a topic of growing interest.

Viscosity acts as another important physical and chemical parameter in cells. It can reflect the status and behaviours of cells and organelles.17–19 In most eukaryotic cells, lysosomes are the main degradative location. They provide essential functions for cellular homeostasis, and impairments in lysosomes are closely associated with continuously increasing numbers of pathological conditions.20–23 Thus, it is essential to evaluate the state of lysosomes in real time. Local hypoxia and viscosity could serve as indicators of lysosome function. Some evidence has been demonstrated where abnormal hypoxia or viscosity is related to some diseases such as hypertension, infection, and inflammation,24,25 although the detailed mechanisms and interactions of either in lysosomes is still not fully understood. Lacking molecular tools for simultaneously tracing the fluctuations of hypoxia and viscosity in lysosomes is an important reason. Therefore, the building of effective tools is urgently needed.

In recent years, compared with traditional detection methods, fluorescent probes have become indispensable tools due to their advantages such as in situ and non-invasive detection, and high sensitivity and selectivity.26–29 Probes for independently detecting NTR and viscosity in lysosomes have been reported, respectively.30–34 The detection of multiple analytes by single-molecule fluorescent probes can effectively avoid the problems caused by the combination of multiple fluorescent probes, such as complex operation and signal crosstalk.35,36 Nonetheless, a single molecular fluorescent probe that can detect both nitroreductase (NTR) and viscosity in lysosomes has been rarely reported.

Here, we present a novel fluorescent probe, NVP, which can visualize the viscosity and NTR in lysosomes simultaneously (Scheme 1). 3-Ketone-4-hydroxy-7-(diethylamino) coumarin was selected as the basic fluorophore owing to its high quantum yield and good photostability. The p-nitrobenzene group is the NTR responsive site. This group is coupled with a π-bridge to construct a sort of D–π–A–π–A frame in the NVP probe. The probe emits no fluorescence owing to the PET effect of the nitro group (–NO2) and twisted intramolecular charge transfer (TICT) effects. However, in high viscosity environments, these rotation effects can be suppressed, resulting in large increases in the fluorescence of the coumarin portion when excited at 380 nm. Furthermore, when encountering NTR, the nitro group (–NO2) of the NVP probe can be changed into the amino group (–NH2), whereupon PET is turned off and the resulting NVH probe emits distinct fluorescence when excited at 440 nm. Notably, in the presence of high viscosity and NTR conditions, the NVH probe can show stronger fluorescence than that when only in the presence of NTR. The synthetic routes are shown in Scheme S1† and the probes are also characterized by 1H NMR, 13C NMR and HRMS in the ESI.†


image file: d4an00906a-s1.tif
Scheme 1 Structure and detailed response mechanisms of the NVP probe towards NTR and viscosity.

2. Results and discussion

2.1 Optical properties of the NVP probe towards viscosity and NTR

With the probe in hand, we first investigated the optical properties of the NVP probe towards viscosity or NTR. For viscosity response testing, mixtures containing different ratios of methanol/glycerol were selected as testing buffers. For NTR response testing, PBS buffer (10 mM, pH 7.4) was chosen as the testing buffer. As shown in Fig. S1,† the NVP in mixtures of different viscosity showed two distinct absorption peaks at 360 nm and 435 nm. The NVP probe (10 μM) exhibited almost no fluorescence in pure methanol when excited at 380 nm (Fig. 1a), while with buffers containing gradual increases of glycerol (0–100%, v/v), fluorescence peaks with the probe became accordingly much stronger with around 20-fold signal enhancement at 457 nm in 100% v/v glycerol. In addition, the fluorescence intensity (log[thin space (1/6-em)]I457) showed a good linear relationship with the viscosity (log[thin space (1/6-em)]η) (R2 = 0.99) (Fig. 1b). Besides, compound 2, the coumarin derivative, showed notable fluorescence accompanied by increases of viscosity in Fig. S2,† indicating that the free rotor between diethylamino and the coumarin precursor had been supressed in compound 2 and NVP. These results demonstrated that the probe has distinct performances with respect to viscosity.
image file: d4an00906a-f1.tif
Fig. 1 (a) Fluorescence spectra of 10 μM NVP in viscosity buffer containing various fractions of methanol and glycerol (0–100%, v/v), λex = 380 nm. (b) The relationship between variation of the system viscosity log[thin space (1/6-em)](η) values and log[thin space (1/6-em)](I457) values. (c) Fluorescence spectra of NVP (10 μM) with NTR (0–0.9 U mL−1) coexisting with 100 μM NADH in PBS buffer (10 mM, pH = 7.4), λex = 440 nm. (d) The linear relationship between I543 and various NTR concentrations (0–0.9 U mL−1).

Then we investigated the behaviours of the NVP probe (10 μM) when detecting NTR in PBS buffer (10 mM, pH = 7.4). There was an increasing absorption signal centered at 450 nm with increasing concentration of NTR (Fig. S3†). Then the fluorescence behaviours of the NVP probe towards NTR were investigated. The NVP probe displayed almost no fluorescence in the presence of NADH (100 μM) when excited at 440 nm, while an obvious fluorescence peak appeared at 543 nm after the addition of NTR (0–0.9 U mL−1) (Fig. 1c) with the fluorescence emission signal of the probe being obviously enhanced (8.6-fold). Besides, there was an admirable linear relationship between the fluorescence signal (I543) and concentration of NTR (0.00148–0.9 U mL−1, R2 = 0.99) (Fig. 1d) and the limit of detection (LOD) was measured as 0.00148 U mL−1 according to the 3σ/k method, which confirmed that NVP has ultra-sensitivity to NTR.

Subsequently, the time-dependent fluorescence signal changes of NVP with NTR concentration (0.3, 0.6 and 0.9 U mL−1) were observed (Fig. 2a). By observing the three curves, it could be seen that when the concentration of NTR was fixed, the fluorescence intensity (I543) gradually increased over time, and reached stability after around 32 min. Besides, the fluorescence intensity of NVP did not fluctuate in the presence or absence of NTR after further irradiation for 30 min. These data indicate that the probe has excellent optical stability. Based on the perfect response results of NVP to viscosity and NTR, we further investigated whether there were unexpected changes in the fluorescence spectra in response to NTR under viscosity conditions (70% glycerol). We found that a 1.67-fold enhancement of fluorescence intensity at 543 nm was obtained in response to NTR in 70% glycerol buffer when compared to that with PBS buffer (Fig. 2b). These results prove that the probe could achieve responses to NTR under high viscosity conditions.


image file: d4an00906a-f2.tif
Fig. 2 (a) Time-dependent fluorescence intensity (I543) changes of 10 μM NVP with various concentrations of NTR (0.3 U mL−1, 0.6 U mL−1 and 0.9 U mL−1) combined with 100 μM NADH within 60 min. (b) Fluorescence response spectra of 10 μM NVP towards NTR (0–0.9 U mL−1) in viscosity buffer (70% glycerol).

It is well known that inside the biological environment it is very complex; therefore, the anti-interference ability of the probe determines whether the probe is suitable for cellular imaging to some extent. So, we examined the ability of the probe to respond to some relevant analytes in biological systems. During experiments, many relevant interfering analytes, such as bioenzymes, biothiols, amino acids, anions, reactive oxygen species and glycerol, were treated with NVP (containing 100 μM NADH) when excited at 440 nm for 40 min, and fluorescence values were measured. As shown in Fig. S4,† it can be found that only the solution with the addition of glycerol has significant fluorescence at the excitation wavelength of 380 nm and has obvious fluorescence in the presence of NTR at excitation of 440 nm, while the fluorescence changes of solutions containing other related analytes were almost negligible. Therefore, these results indicate that NVP can effectively respond to both viscosity and NTR.

Next, the reaction mechanism of the NVP probe towards NTR was further studied. With reference to previous work, the structure of NVP was confirmed reasonably, therefore the product of the reaction between the NVP probe and NTR was obtained. From the spectrum shown in Fig. S6,† the results from high-resolution molecular spectroscopy analysis at m/z = 457.1373 confirmed the existence of NVP. In addition, a peak appeared at m/z = 405.1808 corresponding to NVH ([M + H]+ = 405.1809). This phenomenon is attributed to release of NVH in the reaction. Thus, the proposed sensing mechanisms of NVP with NTR were reasonably in line with those shown in Scheme 1.

2.2 Co-localization imaging of the NVP probe in living cells

The excellent spectral performances of the NVP probe encouraged us to explore its ability to visualize viscosity and NTR in HeLa cells. Before imaging, the cytotoxicity of NVP on HeLa cells was firstly assessed using a CCK-8 kit. The survival rates of cells was more than 85% after incubation with NVP (0–40 μM) for 24 h (Fig. S5†). This demonstrates that the NVP has less cytotoxicity to living cells and is suitable for imaging in living cells. Next, we performed co-localization experiments under hypoxic conditions with HeLa cells to enhance the probe fluorescence so that we could observe the distributions of NVP probes in cells. The distinct green fluorescence from NVP was observed in cells after incubation with 2% O2 for 6 h (Fig. 3a). Signals overlapped well with the red fluorescence from the cells stained with Lyso-tracker™ deep red (Fig. 3b and c), and the trends of the fluorescence signals of the green and red channels located in the region, as indicated by arrows, were almost identical, and the Pearson coefficient was as high as 0.87 (Fig. 3d and e). Therefore, NVP is able to concentrate well in the lysosomes in HeLa cells.
image file: d4an00906a-f3.tif
Fig. 3 Co-localization imaging in HeLa cells between the NVP probe and Lyso deep red. (a) Image from the green channel of the NVP probe, λex = 488 nm, λem = 500–550 nm. (b) Image from the Cy5 channel of Lyso Tracker™ deep red (Lyso deep red), λex = 647 nm, λem = 663–740 nm. (c) The merged image of groups (a) and (b). (d) Intensity scatter plot of the green and Cy5 channels. (e) Marked intensity profile changes of NVP and Lyso deep red. Scale bar: 20 μm.

2.3 Cellular imaging with the treatment of chloroquine using the NVP probe

According to spectrum tests, both fluorescence emission peaks of the probe when detecting viscosity and NTR can be clearly distinguished under different excitation wavelengths, which is conducive to dual-channel imaging. Subsequently, we tested its potential for viscosity detection in HeLa cells. We utilized dual-channel mode in the observation of viscosity and NTR in HeLa cells, respectively. According to the spectral data, the blue channel (λex = 405 nm, λem = 425–475 nm) was set for the detection of viscosity. At the same time, viscosity and NTR were detected by the green channel (λex = 488 nm, λem = 500–550 nm). Chloroquine (Chlor), a common lysosomal inhibitor, inhibits lysosome enzyme activity and reduces lysosomal acidity and changes lysosome viscosity. The corresponding results are displayed in Fig. 4, where cells treated only with the NVP probe (10 μM) emit no fluorescence in the blue channel when excited at 405 nm. However, cells pre-treated with Chlor (30 μM) for 1 h or 2 h, and then incubated with NVP (10 μM) emitted strong blue fluorescence, and this fluorescence increased with the extension of time. These phenomena suggest that the enhancement of fluorescence may be attributed to increases of lysosome viscosity induced by chloroquine.
image file: d4an00906a-f4.tif
Fig. 4 Cellular fluorescence imaging of NVP (10 μM) in living cells. (a) Cells incubated with 10 μM NVP for 30 min; (b) cells were pre-incubated with Chlor (chloroquin, 30 μM) for 1 h, then treated with 10 μM NVP for 30 min; (c) cells were pre-incubated with Chlor (30 μM) for 2 h, then treated with 10 μM NVP for 30 min; (d) mean fluorescence intensities of groups (a), (b), and (c). Blue channel: λex = 405 nm, λem = 425–475 nm; green channel: λex = 488 nm, λem = 500–550 nm. Scale bar: 20 μm.

2.4 Cellular imaging using the NVP probe under hypoxia conditions

NTR is highly expressed in hypoxic cancer cells, so HeLa cells were incubated with 20% O2 (normoxic conditions), and 5% O2 and 2% O2 (hypoxic conditions) for 6 h, respectively, and then further treated with 10 μM NVP for 30 min. As shown in Fig. 5, the cells cultured with 20% O2 exhibited almost no fluorescence in the green channel. The cells stained under hypoxia conditions showed strong fluorescence, and the fluorescence under 2% O2 conditions was brighter than that under 5% O2 conditions, which indicates that the NVP probe has responded to NTR and the concentration of NTR in cells was proportional to the degree of hypoxia. Therefore, these results indicate that NVP can detect NTR in living cells under conditions of hypoxia.
image file: d4an00906a-f5.tif
Fig. 5 Cellular fluorescence imaging of NVP (10 μM) in HeLa cells. (a) Cells were incubated under normoxic conditions (20% O2) for 6 h, and further treated with NVP (10 μM) for 30 min; (b) cells were incubated under hypoxic conditions (5% O2) for 6 h, and then treated with NVP (10 μM) for 30 min; (c) cells were incubated with 2% O2 for 6 h, and then treated with NVP (10 μM) for 30 min; (d) mean fluorescence intensities of groups (a), (b), and (c). Green channel: λex = 488 nm, λem = 500–550 nm. Scale bar: 20 μm.

2.5 Cellular imaging with the treatment of dicoumarin using the NVP probe

In order to verify that the fluorescence changes in the green channels originated from the expression of NTR, dicoumarin, a kind of well-known NTR inhibitor was used.37 During bioimaging, HeLa cells were first cultured at 2% O2 for 6 h, then pre-treated with 100 μM dicoumarin for 1 h, and finally incubated with 10 μM NVP for 30 min. The fluorescence of the green channel in the dicoumarin-treated cells was significantly reduced compared to that of the untreated cells. Thus, it is clear that the increased fluorescence of lysosomes in HeLa cells under hypoxia conditions is due to the response of NVP to NTR. Therefore, the NVP probe can be used to monitor the dynamic changes of lysosomal NTR (Fig. 6).
image file: d4an00906a-f6.tif
Fig. 6 Cellular fluorescence imaging of NVP (10 μM) in HeLa cells. (a) Cells incubated with 10 μM NVP probe under normoxic conditions (20% O2) for 30 min; (b) cells were incubated under hypoxic conditions (2% O2) for 6 h, and then treated with NVP (10 μM) for 30 min; (c) cells were cultured under 2% O2 hypoxia conditions for 6 h, followed by treatment with 100 μM dicoumarin for 30 min and then treated with NVP (10 μM) for 30 min; (d) mean fluorescence intensities of groups (a), (b) and (c). Green channel: λex = 488 nm, λem = 500–550 nm. Scale bar: 20 μm.

2.6 Cellular imaging with the treatment of chloroquine under hypoxia conditions using the NVP probe

According to the above results, the NVP probe can detect both viscosity and NTR, respectively. We further investigated whether the NVP probe was suitable in responding to NTR under changes of viscosity. HeLa cells were treated under hypoxic conditions (2% O2) for 6 h, then incubated with chloroquine for 1 h, and finally incubated with NVP for 30 min. As shown in the Fig. 7, a significant enhancement of the fluorescence signal was observed in the green channel when comparing to cells treated under conditions of hypoxia and chloroquine alone. These results proved that the NVP probe could be used to detect NTR in lysosomes of living cells under viscosity conditions. Taken together, these results were consistent with the spectral results, indicating that NVP can track NTR and viscosity changes not only in lysosomes but also under viscosity conditions.
image file: d4an00906a-f7.tif
Fig. 7 Fluorescence imaging of NVP (10 μM) in HeLa cells. (a) Cells incubated with the free NVP probe (10 μM) under normoxic conditions (20% O2) for 30 min; (b) cells were incubated under hypoxic conditions (2% O2) for 6 h, and then treated with NVP (10 μM) for 30 min; (c) cells were cultured under 2% O2 hypoxia conditions, followed by treatment with 30 μM Chlor for 1 h and then treated with NVP (10 μM) for 30 min; (d) mean fluorescence intensities of groups (a), (b) and (c). Green channel: λex = 488 nm, λem = 500–550 nm. Scale bar: 20 μm.

2.7 Zebrafish imaging with the treatment of chloroquine using the NVP probe

The NVP probe has achieved good responses towards viscosity and NTR at the cellular level. Next, we investigated the feasibility of NVP imaging viscosity in larval zebrafish. As shown in Fig. 8, zebrafish pretreated with 30 μM chloroquine (1 h and 2 h) showed more obvious fluorescence in the blue channel than those treated without chloroquine. These results are consistent with in vitro data and successfully realized the detection of viscosity in larval zebrafish.
image file: d4an00906a-f8.tif
Fig. 8 Fluorescence images of viscosity in zebrafish cells. (a) Zebrafish were treated with NVP (10 μM) for 30 min; (b) zebrafish were pre-incubated with Chlor (chloroquin, 30 μM) for 1 h, then treated with 10 μM NVP for 30 min; (c) zebrafish were pre-incubated with Chlor (chloroquin, 30 μM) for 2 h, then treated with 10 μM NVP for 30 min; (d) mean fluorescence intensities of groups (a), (b) and (c). Blue channel: λex = 405 nm, λem = 425–475 nm.

2.8 Zebrafish embryos imaging under hypoxia conditions using the NVP probe

We further verified changes of NTR levels in zebrafish embryos before and after hypoxia treatment. Zebrafish embryos treated with and without hypoxia were selected as experimental models. First, the embryos were cultured using different oxygen contents (20%O2, 5%O2, 2%O2) for 6 h, and then 10 μM NVP was added to incubate the embryos for 30 min. As shown in Fig. 9, the embryos treated with hypoxia showed obvious fluorescence in the green channel, indicating that the level of NTR in the embryos treated with hypoxia was significantly higher than that in the embryos treated without hypoxia. Therefore, NVP can successfully image the changes in the activity of NTR in the embryos, realizing the visual detection of NTR in the embryos.
image file: d4an00906a-f9.tif
Fig. 9 Imaging of NTR using NVP in zebrafish embryos. (a) Zebrafish embryos were incubated under normoxic conditions (20% O2) for 6 h, and then treated with NVP (10 μM) for 30 min; (b) zebrafish embryos were incubated under hypoxic conditions (5% O2) for 6 h, and then treated with NVP (10 μM) for 30 min; (c) zebrafish embryos were incubated under 2% O2 for 6 h, and then treated with NVP (10 μM) for 30 min; (d) mean fluorescence intensities of groups (a), (b) and (c). Green channel: λex = 488 nm, λem = 500–550 nm.

3. Conclusions

In conclusion, we have designed a single-molecule fluorescent probe for simultaneously visualizing viscosity and NTR in lysosomes based on dual emission channels. The spectral test results have shown that the NVP probe can emit strong fluorescence peak signals at 457 nm with excitation at 380 nm under high viscosity conditions; meanwhile, the probe showed marked peak signals at 543 nm when excited at 440 nm when encountering NTR. Furthermore, the NVP probe has high sensitivity to NTR (the LOD was calculated to be 0.00148 U mL−1). In addition, NVP also has high selectivity and good photostability. These advantages help NVP realize visual detection of viscosity and NTR in biological systems. In terms of bioimaging, the NVP probe has displayed excellent lysosome targeting. NVP has achieved detection of the fluctuations of viscosity in chloroquine-stimulated cells and NTR in hypoxia-stimulated cells with dual-channel responses. Besides, NVP successfully detected viscosity changes in zebrafish induced by chloroquine and the activity changes of NTR in zebrafish embryos under hypoxia conditions. We believe that the probe can be used as an effective tool to detect viscosity and NTR-related diseases, and can provide a theoretical basis for the design of other similar two-site fluorescent probes.

Author contributions

Jingchao Wang: conceptualization, data curation, formal analysis and investigation, methodology, software, writing – original draft. Lina Zhou: data curation, formal analysis, writing – original draft. Zekun Jiang: data curation, formal analysis, software. Haiyan Wu: conceptualization, writing review & editing. Xiuqi Kong: funding acquisition, supervision, writing – review & editing.

Ethical statement

Zebrafish (Danio rerio, 7 days post fertilization) and embryos (1 day post fertilization) were obtained from the School of Pharmaceutical Sciences, Shandong University. They were cultured under the following conditions: 28.5 °C, with a 12 h: 12 h light: dark cycle. Zebrafish were fed twice a day with brine shrimp, and embryos were kept in fish-rearing water. During the experiments, all animal procedures were approved by the Animal Ethical Experimentation Committee of Shandong University and performed strictly in accordance with the “Guiding Principles for Research Involving Animals and Human Beings”.

Data availability

No additional data are available.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was financially supported by the Natural Foundation of Shandong Province (ZR2023MB022) and the National Natural Science Foundation of China (NSFC, 21807039).

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Footnotes

† Electronic supplementary information (ESI) available: Characterization data and additional spectra. See DOI: https://doi.org/10.1039/d4an00906a
‡ These authors contributed equally to this work.
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