A novel NBD-based pH “on–off” fluorescent probe equipped with the N-phenylpiperazine group for lysosome imaging

Abstract

A novel PET-based fluorescent probe (LN6) targeting to lysosome was found from the synthesized NBD derivatives. This probe was equipped with the novel lysosome-targeted N-phenylpiperazine group, and could be employed to image lysosome and detect its pH change without any washing steps.

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As the digestive organelle of cells, lysosome plays critical roles in the biological processes of phagocytosis, endocytosis, and autophagy.^1–3 This organelle exhibits an acidic pH range of 4.0–6.0, and this acidic property provides a favourable environment for interior digestive enzymes to degrade macromolecules and cell components.^4–6 Abnormal lysosomes are associated with various pathological activities, such as cell apoptosis, oncogenic activation and cancer progression.⁷

Since the discovery of lysosome in 1950s, a large study on lysosome has allowed the expansion of tools to understand the cell biology of lysosome, especially the development of the fluorescent probes used in the no-invasive cell imaging.⁸ Over the years, various fluorescent probes have been reported and found widespread application for the visualization of lysosome in living cells.^9,10 In general, due to the acidic environment in lysosome, most of small-molecule probes for lysosome are designed by introducing a basic side chain to a fluorophore, in which the basic side chains would promote the molecules to concentrate in acidic organelle selectively by the protonation effect.^10–12 Additionally, the photoinduced electron transfer (PET) effect generated between some amino side chain, such as 4-(2-hydroxyethyl)morpholine and N,N-dimethylethylenediamine and chromophore, has been used to design the “off–on” probes of lysosome with high sensitivity and high signal to noise (S/N) ratio.^13,14 For example, as shown in Fig. 1, the four molecules are designed on basis of the PET effect of BODIPY or naphthalimide fluorophore, and some of them are commercially available now.^13–18 However, the application of these probes was limited by the difficulty in synthesis, such as instability of fluorophore and tedious synthetic steps. Besides the two fluorophores above, 4-nitro-2,1,3-benzoxadiazole (NBD) is another representative chromophore with a strong intramolecular PET effect.^19,20 Compared with other chromophore derivatives, NBD moiety shows no fluorescence itself, so it may bring a little background signal for imaging research.^21,22 Although several studies have declared that the NBD derivatives could accumulate in acidic vesicles, none introduced the application of NBD derivatives in lysosomal imaging systematically.^23,24

Fig. 1 The reported PET-based lysosomal probes and design strategy of novel NBD-based fluorescent probes.

In this work, according to the design strategy of PET-based lysosomal probes, we designed a series of NBD compounds with different basic side chains including dimethylamine, morpholine and N-phenylpiperazine groups (Fig. 1). As shown in Scheme 1, a total of seven compounds were synthesized through the one-step nucleophilic substitution reactions of 4-chloro-7-nitrobenzoxadiazole (1) and the corresponding side chains (2–8). Thereinto, NBD-Cl (1) and compounds 2–5 could be available commercially, and compounds 6–8 were facilely synthesized from the typical Gabriel reactions as shown in Scheme S1.† Compared with fluorescent probes reported previously, these NBD compounds could be synthesized very facilely.

Scheme 1 Synthesis of compounds LN1–7.

The spectroscopic properties measured in the Britton–Robinson buffer solutions (pH = 7.4), indicated that these compounds (40 μM) display absorption and emission spectra at about 475 nm and 545 nm, respectively, and the Stokes shift is about 70 nm (Fig. 2 and Table 1). As expected, these compounds could emit green fluorescence under the blue excitation light, which would avoid the disturbance of auto-fluorescence from cells or tissues. Moreover, as depicted in Fig. 2b, the seven compounds revealed a major difference in the aspect of fluorescence intensity. At the condition of pH 7.4, compounds LN3, LN5, and LN6 showed weak fluorescence, while other four compounds' fluorescence was strong relatively. Additionally, the fluorescence quantum yields of compounds were measured (Table 1). These compounds gave very different quantum yields as well as the different fluorescence intensity. For these compounds, the major difference in chemical structure among them is from the side chains. Therefore, the difference in fluorescence intensity and quantum yield among them should be caused by the different side chains. These results above indicated that the compounds with longer side chain possessed stronger fluorescence or higher quantum yield.

Fig. 2 Absorption and emission spectra of compounds LN1–7.

Table 1 Data about spectroscopic properties of compounds LN1–7

Compds	λex/nm	λem/nm	Φ^a/%	IC50^b/μM
a 10 mM PBS solution, pH = 7.4.b Cytotoxicity on HeLa cells.
LN1	465	545	2.61	>100
LN2	475	545	3.77	>100
LN3	480	545	0.44	>100
LN4	470	545	1.60	>100
LN5	480	550	0.04	>100
LN6	475	545	0.79	>100
LN7	475	545	1.88	>100

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To further understand the PET effect of compounds, we examined thoroughly their fluorescence response to acid–base environment. As displayed in Fig. 3, fluorescence of each compound showed different behaviours following the change of pH values. For compounds LN1, LN3, LN4, LN5 and LN6, the fluorescence intensity presented an S curve change, and in detail, their fluorescence was strong in acidic solutions, diminishing in the leaning neutral condition, and weak in basic environment. This type of curves here are highly consistent with the situation of the reported lysosomal probes in Fig. 1, indicating that the change may be attributed to the intramolecular PET effect between the side chain and NBD fluorophore.^15,25 In principle, at non-acidic conditions, the amine-containing side chains can quench fluorescence of dyes via PET effect, while in acidic environment the amino groups will be protonated to allow the fluorescence to release. Relatively speaking, the response of LN2 and LN7 to pH values showed unobvious change in emission intensity, which may be related to their weak PET effect caused by their relatively longer linker. According to the pK_a values for LN1 (6.81), LN3 (5.42), LN4 (6.98), LN5 (5.17) and LN6 (5.72), which were calculated based on the S curves and the Henderson–Hasselbalch equation, it could be demonstrated that LN3, LN5 and LN6 can detect the pH change in 5.0–6.0 range, which is suitable for the acidic lysosome.²⁶ However, the fluorescence of LN3 in basic solutions (quenching state) is relatively strong, which means strong background noise, and the emission of LN5 in acidic condition is too weak representing a weak positive signal. By contrast, LN6 performs the best because there is a more than 30-fold increase in its emission intensity when the pH value changes from 7.4 to 4.0. Its fluorescence is steady in the two large pH ranges of 3.2–5.4 (bright) and 6.6–10.0 (weak), while in the short range of 5.4–6.6, the fluorescence intensity changes sharply. Therefore, this preliminary result declares that LN6 could serve as a pH-tunable probe. Additionally, after being illuminated continuously by the ultraviolet at 365 nm, LN6 emitted almost changeless fluorescence intensity, showing this compound has good photostability and high value for real-time monitoring (Fig. S1†).²⁵

Fig. 3 Changes in fluorescence intensity of compounds LN1–7 in Britton–Robinson buffers at different pH values (3.2–10.0). In the measurement, the excitation and emission wavelengths for each compound were consistent with the values in Table 1.

We next investigated the effect of these compounds on HeLa cell viability and the result indicated that these NBD derivatives had no cytotoxicity at the concentration of 100 μM (Table 1). Therefore, we further employed all compounds (50 μM) in the cell imaging research. As shown in Fig. S2,† after incubation for 1 h, LN1, LN2, LN4 and LN7 revealed low level of dyeing, while the cells treated by LN3, LN5 and LN6 showed stronger emission. These behaviours of the latter three compounds may be related with their suitable pK_a values. To confirm if LN3, LN5 and LN6 could target to lysosome, the commercial Lyso Tracker Red (LT Red) was added into cells for co-staining. As a result, we found that there was more non-specific binding existing between LN5 and the cell components, while the localization of LN3 and LN6 in the cytoplasm was consistent with that of LT Red indicating LN3 and LN6 could serve as the lysosomal fluorescent probes (Fig. 4). Recently, compound LN3 named NBDlyso by Cao et al., has just been report as the fluorescent probe for lysosome.²⁵ Herein, except for reaching the similar conclusion for LN3, we found that compared with LN3, LN6 could visualize lysosome with brighter fluorescence and higher S/N ratio, which may be resulted from its more appropriate fluorescence response to pH values. From imaging results above, we found the classical lysosome-targeted groups, including dimethylamine and morpholine groups, work worse than the N-phenylpiperazine group in lysosomal visualization, so it could be speculated that N-phenylpiperazine moiety maybe a novel lysosome-targeted group.^27,28 This conclusion could help researchers understand the cellular distribution of N-phenylpiperazine-containing compounds.

Fig. 4 Confocal images of HeLa cells that were co-stained by LT Red and three compounds together. Cells were incubated with LN3, LN5 or LN6 (50 μM) for 1 h at 37 °C and then the probe solutions were discarded followed by LT Red was added to incubate for another 30 min. After washing with PBS buffer, the cell imaging was carried out on the LSM 700 or 780 microscope. Green: the fluorescence of probe compounds (λ_ex = 488 nm); red: the fluorescence of LT Red (λ_ex = 594 nm). Objective lens, 63×.

Compound LN6 is a powerful pH “on–off” probe, and this compound could target to the lysosomes in the living cell. The dual properties of LN6 may bright great benefits to visualization of lysosome. In the following study, we found it is satisfying to employ LN6 in the no-washing lysosome imaging. HeLa cells were firstly incubated for 30 min at 37 °C with LT Red, Mito Tracker Red (MT Green) and Golgi Tracker Red (GT Red), respectively, and following discarding the tracker solutions, LN6 solutions (50 μM) were added to incubate for another 60 min. Subsequently, the cells were recorded on the confocal microscope without any washing. The images in Fig. 5 demonstrated that the localization of LN6 on the cells was nearly identical to that of LT Red (Fig. 5A), but did not overlap with that of MT Red and GT Red (Fig. 5B and C). We further calculated the Pearson's colocalization coefficient using the ImageJ software to evaluate the colocalization level of LN6 and the three commercial trackers.²⁹ The coefficient of LT Red is up to 0.83, which is higher than that of MT Red (0.38) and GT Red (0.55). Additionally, if the incubation time was prolonged, the colocalization coefficient of LN6 and LT Red would increase partly (0.91, Fig. 5D). Therefore, we can draw a conclusion that LN6 could visualize lysosomes in living cells specifically even without any washing steps.³⁰

Fig. 5 Co-localization of LN6 with LT Red (A and D), MT Green (B) or GT Red (C). Cells were incubated with LN6 for 60 min (A–C) or 24 h (D). (a) Bright-field images; (b) cells stained by LN6 (λ_ex = 488 nm); (c) cells stained by LT Red (Ac and Dc, λ_ex = 594 nm), MT Red (Bc, λ_ex = 594 nm) or GT Red (λ_ex = 594 nm); (d) merged images. Objective lens, 63×.

In general, abnormal lysosomes are associated with the increase of their pH values. Some basic substances, such as chloroquine, can cause alkalization of lysosome through the leakage or neutralization of lysosomal proton.³¹ Considering that LN6 is a pH-tunable probe, it was used to detect the chloroquine-induced lysosomal pH changes in the next study. As shown in Fig. 6, LN6 emitted bright fluorescence in the normal HeLa cells treated without chloroquine, however, when a mass of chloroquine was added to stimulate cell beforehand, the fluorescence on cells decreased clearly. Furthermore, the large concentration of chloroquine (100 mM) could weaken the fluorescence intensity of LN6 more significantly than the low concentration of chloroquine (25 mM). This result indicated that LN6 could be employed as a pH-responsive fluorescent sensor for lysosome imaging in living cells.

Fig. 6 Fluorescence images of HeLa cells treated by LN6 together with or without chloroquine. (A) Cells were incubated with 50 μM of LN6 only; (B) cells were incubated by 25 mM of chloroquine for 30 min firstly, followed by the 1 h-incubation with 50 μM of LN6; (B) cells were incubated by 100 mM of chloroquine for 30 min, followed by 1 h-incubation with 50 μM of LN6. (a) Images in the bright field; (b) images in green fluorescence channel; (c) merged images of (a) and (b). Objective lens, 63×.

Conclusions

In conclusion, based on the NBD fluorophore with typical PET effect, we designed and synthesized a series of NBD derivatives by introducing the side-chain dimethylamine, morpholine or N-phenylpiperazine groups. Among these compounds, LN3 with the morpholine group and LN6 with N-phenylpiperazine groups displayed appropriate PET response, which could be utilized in lysosome imaging. Significantly, compared with LN3, LN6 could produce stronger emission to visualize lysosome clearer due to its more sensitive pH response. LN6 also showed a little cytotoxicity and good photostability. Further study indicated LN6 could be employed in the no-washing cell imaging of lysosome and the detection of chloroquine-induced pH changes in lysosome. Thus far, we have developed a novel pH “on–off” fluorescent sensor for lysosome imaging, which is equipped with a new-found lysosome-targeted N-phenylpiperazine group. This probe could be synthesized facilely from readily available starting materials and can exhibit green fluorescence with large Stokes shift and high S/N ratio.

Acknowledgements

The present work was supported by grants from the Qilu Professorship at Shandong University, the International Postdoctoral Exchange Fellowship Program, the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13028), the Major Project of Science and Technology of Shandong Province (No. 2015ZDJS04001), the Shandong Key Research & Development Project (No. 2015GSF118166) and the Fundamental Research Funds of Shandong University (No. 2014JC008). Our cell imaging work was performed at the Microscopy Characterization Facility, Shandong University.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra16996a

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