An aggregation-induced emission-based pH-sensitive fluorescent probe for intracellular acidity sensing

Abstract

A dual-emission pH-sensitive fluorescent probe was developed, which displays green fluorescence in alkaline after deprotonation, while orange emission in acid at aggregated state. This novel probe allows the specific light-on in acidic lysosomes of cancer cells and tumors in nude mice, which indicates its potential application in cancer diagnosis.

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Intracellular pH (pH_i) plays an essential role in most regulating cellular behaviours, such as vesicle trafficking, cellular metabolism, proliferation and apoptosis.¹ As a typical example, a number of cellular metabolic pathways, including endocytic processes, signalling, apoptosis or defence, are regulated by the pH_i value changing to be in the acidic range, as an acidic environment helps to activate enzyme functions and protein degradation.² Therefore, visualization of these important events will allow us to gain better insight into many physiological or pathological processes, such as cancer diagnosis.³ To date, pH_i measurements include proton-permeable microelectrodes, NMR spectroscopy and fluorescence imaging.⁴ Among these techniques, fluorescence-based techniques might be the most powerful tools for the observation of pH_i changes, due to its high sensitivity and outstanding spatio-temporal resolution.^4c The design of fluorescent probe for pH_i measurement is mostly based on proton binding, photo-induced electron transfer (PET), excited-state intramolecular proton transfer (ESIPT), fluorescence resonance energy transfer (FRET) and so forth as the working principles at present.^4c,5 In order to further investigate the essential roles of acidity in cellular condition, it is highly desire to develop other strategies for designing pH_i fluorescent probes.

The discovery of fluorophores with aggregation-induced emission (AIE) characteristics has changed the way of thinking and opened new opportunities to develop light-up probes for bio-sensing, imaging and therapy.⁶ These fluorophores are non-emissive in the molecularly dissolved state but induced to emit strong fluorescence at aggregated state due to the restriction of intramolecular rotation (RIR) and prohibition of energy dissipation through nonradiative routes.^6a,b A great variety of AIE based chemosensors and biosensors have been developed for different targets so far.⁷ For example, a pH sensitive AIE molecule (TPE-Cy) has been developed for intracellular pH sensing based on the combination of AIE active unit-TPE with pH-sensitive dye-hemicyanine.⁸ The preparation of TPE-Cy desired complicated process with multiple steps. Recently, a facile strategy to construct AIE-based pH fluorescent probe was based on Schiff base. For example, few salicylaldehyde Schiff base azines have been used as pH sensors for cell imaging.⁹ However, these pH sensors only are used in cell imaging, the application for in vivo imaging especially for tumor tissue has not been investigated.

In this paper, we reported a dual emission pH-sensitive AIE probe, as environmental pH value could easily affect the structure and solubility of this unique molecule containing carboxyl and hydroxyl groups. This AIE probe which underwent deprotonation emitted green fluorescence at 495 nm at weak alkaline solution, while the aggregated form of the probe in acid medium displayed orange fluorescence at 590 nm. The pH-sensitive mechanism was investigated by means of UV-visible and NMR spectroscopic techniques. Moreover, the AIE probe has been successfully applied for fluorescent visualization of intracellular acidity owing to its unique properties, such as excellent cell membrane permeability, high stability and good reversibility.

This novel fluorescent probe (4,4′-(hydrazine-1,2-diylidene bis(methanylylidene)) bis(3-hydroxybenzoic acid), named HDBB) was synthesized from 4-formyl-3-hydroxybenzoic acid by a facile one-step condensation reaction and obtained in a reasonable yield with 73.5% (shown as in Fig. 1A). The detailed synthetic procedure and characterization data are available in the ESI.‡ Molecular structure was further confirmed by NMR and mass spectroscopy (Fig. S1–S3‡).

Fig. 1 (A) The scheme for synthesis process of HDBB by one-step condensation reaction from 4-formyl-3-hydroxybenzoic acid. FL spectra (B) and plots (C) of FL intensity versus methanol fraction of HDBB in mixtures of DMSO/methanol with different methanol fractions. Dye concentration = 50 μM, excitation wavelength = 365 nm. Inset in (C): the photographs of HDBB in fm = 0 (left) and fm = 90 (right) mixtures of DMSO/methanol upon excitation at 365 nm using an ultraviolet lamp.

Salicylaldehyde hydrazine derivatives have recently attracted great research interest in the areas of biosensing and imaging due to their unique AIE property.⁹ To test whether the HDBB retained the AIE properties, we studied the fluorescence intensity of HDBB in dimethyl sulfoxide (DMSO)/methanol mixtures with different methanol fractions (fm). As shown in Fig. 1B and C, HDBB was almost non-fluorescent as a solution in DMSO (fm = 0). However, as the fm value increased, the fluorescence intensity increased steadily. At fm = 90 vol%, an 82-fold enhancement of orange emission at 590 nm with a large Stokes-shift of 230 nm was observed as compared to that in DMSO, revealing an obvious AIE effect. The increase in fluorescence intensity indicated that HDBB tended to aggregate when the fm value increased, thus resulting in a restriction of the intramolecular motion. The aggregate formation of HDBB was also confirmed by light scattering under normal light (Fig. S4‡). These aggregations were sphere shaped with a uniform diameter of 54.0 ± 3.2 nm from SEM imaging (Fig. S5‡).

More interestingly, the fluorescent emission of HDBB was also sensitive to pH change. The changing fluorescence emission spectra of the probe with varying pH from 2 to 13 were shown in Fig. 2C. The dye was orange luminescent at 590 nm at low pH values and its fluorescent intensity remained almost constant when the pH < 4. At pH > 4, the orange emission faded gradually and completely vanished at pH 7.0. When pH value was above 8.0, a new green emission peak emerged at 495 nm and the fluorescent intensity increased promptly with pH, as showed by the highly bright green fluorescent images in Fig. 2A. The quantum yields and life time of orange and green emission of this probe are 5.32%, 1.56% and 1.97 ns, 2.31 ns, respectively (Fig. S6 and S7‡). The pK_a1 and pK_a2 of HDBB are 5.61 and 9.88, which are calculated by fluorescent method (Fig. S8 and S9‡). Most notably, the pH-sensitive probe displayed an excellent reversibility between alkaline and acid medium (Fig. S10‡). Such response was very fast and can be repeated for many cycles without any change in the spectral profiles. The fast response and high reversibility of this probe will be of great benefit to its further bio-sensing and imaging application.

Fig. 2 The photographs of HDBB in the aqueous solutions with pH ranging from 2 to 13 upon excitation at 365 nm using an ultraviolet lamp (A) and daylight lamp (B). (C) pH dependent fluorescent spectra of HDBB in the aqueous solutions with pH ranging from 2 to 13 measured under excitation of 365 nm. (D) pH dependent UV-visible spectra of HDBB in the aqueous solutions with pH ranging from 2 to 13. Dye concentration = 25 μM.

The absorption spectra of HDBB were also sensitive to pH change (as shown Fig. 2D). With the increase of pH value from 2 to 13, the solutions changed its appearance from colorless to yellow. In acidic buffer solutions, the dye exhibited two absorption peaks at 310 nm and 365 nm, probably ascribed to the π–π* and n–π* transitions of the entire molecule. When the pH was up to 7, a new absorption peak at 425 nm was observed, suggesting the formation of new chemical species. The absorption peaks at 310 nm and 365 nm gradually decreased while counterpart at 425 nm increased with the increasing pH value above 7, indicating the transition of HDBB to the new chemical species. Similar with fluorescent response, the colorimetric transition was also fast and reversible upon pH change(Fig. S11‡).

This novel phenomenon of pH-switched orange/green emission stimulates us to investigate the underlying working mechanism. In this study, there are carboxyl and hydroxyl groups in HDBB molecule. Generally, the protonation/deprotonation (always formed salt) of these groups have an important role in the solubility of the molecule. We proposed that a reversible chemical reaction of the probe with OH⁻/H⁺ should account for such phenomenon. This was supported by the observation of HDBB aggregation in acidic medium by light scattering (Fig. 2B). The optical and chemical properties of HDBB in acidic solution were similar to the one in methanol solution, which are suggesting that the fluorescent orange emission of HDBB in acidic medium is resulted from the AIE effect. Moreover, the ¹H NMR and ¹³C NMR spectra were utilized to investigate the molecular structure changes of HDBB in neutral and alkaline medium, respectively (Fig. 3A and B). The large shift to low field of two carbon signals (h, e in Fig. 3B) could be ascribed to the formation of HDBB salt. This is consistent with high pH induced solubility increase of HDBB (Fig. 2B). The formation of HDBB salt resulted in the increasing electron density in benzene, which renders the proton signals to shift to the high field and the peak splits. This was confirmed by the red shift in UV-visible spectrum of HDBB in alkaline medium (Fig. 2D).

Fig. 3 ¹H NMR (A) and ¹³C NMR (B) spectra of HDBB in neutral and alkaline medium. (C) Protonation and deprotonation structure processes of HDBB in acidic and alkaline medium.

In connection with the dye working mechanism, a scheme can be drawn to understand the pH-sensitive colorimetric and fluorometric transitions of HDBB (Fig. 3C). In acidic medium, HDBB can hardly be dissolved because it contains large hydrophobic moiety. Several HDBB molecules may cluster into stabilized packing aggregates. The RIR process is thus activated by the relative strong intermolecular physical packing, turning on the orange emission. The aggregates become more and more compact with the decreasing pH, which induces the increasing orange fluorescent signals. With the increasing pH value, HDBB is deprotonated to form the salt with green emission in alkaline medium.

Considering that some salicylaldehyde Schiff base derivatives can chelate metal ions in solution,^9b,d the selectivity of HDBB to H⁺ over metal ions was investigated by competition experiments. The relative fluorescence intensity of the probe in the absence or presence of an excess of K⁺, Zn²⁺, Mg²⁺, Ca²⁺, Fe³⁺, Cu²⁺, Mn²⁺ and Cd²⁺ ions at pH = 7.40 and pH = 5.0 are shown in Fig. S14,‡ which indicates that the effect of such metal ions on pH measurement is negligible, particularly when considering that the concentrations used for the experiment were significantly higher than those present in the intracellular environment.

To demonstrate the potential biological application of our novel AIE probe, HepG2 human liver cancer cells as well as nude mice model were utilized to investigate its in vitro or in vivo pH sensing ability. Before test, the viability of HepG2 and HEK293 cells with exposure of HDBB by using in vitro methylthiazolyltetrazolium (MTT) assay were first evaluated and confirmed above 90% cell viability with the exposure of a dose of 0.1 mM for 24 h, suggesting that HDBB had good bio-compatibility (Fig. S15‡). The influence of intracellular species, such as ions, saccharides and proteins, on the quantitative pH determination were investigated, revealing that these species over their physiological concentrations exhibit negligible effect (Fig. S16‡). Then, we incubated HDBB with HepG2 cells for 10 min, and obtained confocal images with co-staining of Lyso Tracker (a commercial fluorescent dye targeting to lysosome), as shown in Fig. 4A. This novel AIE molecule could fast penetrate the cell membrane into intra-cellular cytoplasm, and showed strong localized fluorescence within Lyso Tracker, indicating its specific intracellular location of lysosomes. As lysosome is distinct from other cellular organelles because of its low pH (below 6.0), the unique enhancement of fluorescence signals of HDBB might be due to its AIE character, which agreed well with previous observation in Fig. 2. The pH value of the lysosome in Fig. 4 has been determined as 5.09 by using HDBB as pH indicator (the calculated process is shown in ESI‡).

Fig. 4 (A) In vitro confocal image of HepG2 cells incubated with 20 μM HDBB (orange) for 1 h. Lyso Tracker (red) was co-stained to visualized the location of lysosomes in HepG2 cells. (B) In vivo non-invasive fluorescence imaging of nude mice bearing HepG2 tumor with post intravenous injection of 50 μM HDBB for indicated time points. The black arrow indicated the location of tumor. (C) Ex vivo fluorescence imaging of tumor and major organs isolated from nude mice, sacrificed after 12 h.

As the pH value of lysosomes in tumor might be lower than that in normal tissues as well as tumor excellular environment is more acidic than normal tissues, we intended to investigate the potential of HDBB for cancer diagnosis. For this purpose, nude mice bearing HepG2 tumor were treated with HDBB by intravenous administration and were subsequently imaged by Capiler IVIS Lumina IV Imaging System. The autofluorescence signals from mice were removed by spectral unmixing by using IVIS software. Fig. 4B showed the in vivo distribution of HDBB fluorescence and its specific tumor accumulation in living nude mice. Furthermore, tissues including tumor, heart, liver, spleen, and kidney were isolated to evaluate the tissue distribution of fluorescent signals in Fig. 4C. Experimental results showed that the fluorescence was only observed in tumor but rarely in other organs, indicating that HDBB was non-fluorescence at normal tissues without more acidic environment. In short, HDBB could yield strong fluorescence in lysosomes of cancer cells and even tumor, indicating its potential application for valuable cancer diagnosis.

In conclusion, the preceding results show the success of design and application of a dual emission AIE based pH-sensitive fluorescent probe. Owing to the effect of pH value on the structure and solubility, this probe displays green fluorescence in weak alkaline solution after deprotonation, while orange emissions at aggregated state. In vitro or in vivo imaging studies revealed fluorescent intracellular acidity can be observed by this probe with excellent cell membrane permeability, large Stokes-shift and high stability, which indicates its potential application in cancer diagnosis. This new strategy for designing pH sensitive probe provides an alternative route for the fabrication of luminescent probes and related applications.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (NSFC, 21405135, 21303145), the Project sponsored by SRF for Yunnan university (2013CK006), Cultivation Program for Key Young Teachers of Yunnan University (XT412003) and the Science and Technology Project of Fujian Province (2014J01063).

Notes and references

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Footnotes

† All animal experiments were carried out in accordance with guidelines approved by the Animal Care and Ethics Committee of Xiamen University.

‡ Electronic supplementary information (ESI) available: Detailed synthetic procedure and characterization data. See DOI: 10.1039/c6ra01167e

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