Near-infrared fluorescent probes with higher quantum yields and neutral pK_a values for the evaluation of intracellular pH

Abstract

We have developed two near-infrared fluorescent probes for pH, named pH-A and pH-B. The fluorescence quantum yields of pH-A and pH-B are 0.19 and 0.095 in methanol. And there is the neutral pK_a value of pH-A (pK_a = 7.2). What is the most important is that these probes can stain the cells, which can reflect the changes of pH in cells.

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The varieties of intracellular pH play important roles in various biological processes such as receptor-mediated signal transduction, enzymatic activity,¹ cell proliferation,² apoptosis,³ ion transport,⁴ and homeostasis.⁵ Abnormal pH values are often associated with cell dysfunction and some diseases.^6,7 Thus, monitoring intracellular pH values can provide direct information on living cells.

Fluorescence probes have been widely studied in fluorescence imaging in living cells, tissues and organisms.^8–10 In order to search for a practicable luminescent probe for pH, it may be examined via spectroscopy and microscopy inside living cells. The most important property for the probe is that it needs to be cell-permeable, kinetically stable and non-toxic in living cells. So far, a number of pH probes have been studied and some are commercially available.^11–13 However, many probes have bad permeability of cell membrane, poor stability, low fluorescence quantum yield, short analytical wavelength or lower or higher pK_a than physiological conditions.^12–14 Thus, the development of a new pH probe that has available pK_a and long wavelength is still in demand.

Cyanines, a classic type of near-infrared fluorochromes, have been frequently employed to develop different spectroscopic probes for imaging studies,^14–16 but they are known to have poor stability and low fluorescence quantum yield as a result of ready autoxidation and photooxidation. And rhodamine fluorophores, which have high fluorescence quantum yield, excellent biocompatibility and high sensitivity under physiological conditions, have been used extensively to measure intracellular pH values.¹⁷ They display strong red fluorescence (excitation/emission, 563/572 nm) at an acidic pH because of the opening of their cyclolactam ring, and become non-fluorescent at a basic or neutral pH when the ring is closed.^17,18 In this context, we want to develop pH probes, which combine cyanines and rhodamine into one molecule, and which have contrasting fluorescence emission in response to pH changes, and therefore would be fluorescent probes to measure intracellular pH values.

With these criteria in mind, we have looked up the literature, and studied the properties of some dyes. In 2012, Lin’s group presented a strategy to synthesize near-infrared fluorescent dyes.¹⁹ And we have found that fluorescent dye C has a large absorption extinction coefficient, high fluorescence quantum yield (Φ_f 0.41 in methanol) with a near-infrared emission peak at 728 nm, good photostability and chemical stability. Inspired by the attractive photophysical properties of dye C, we designed and synthesized two near-infrared fluorescent pH probes, named pH-A and pH-B, which have a similar fluorophoric structure. And we speculate that their pK_a is close to neutral and high fluorescence quantum yield. Fortunately, two near-infrared fluorescent pH probes have been obtained. And the probes have good permeability of cell membrane, long analytical wavelength and available pK_a and low toxicity in living cells. And as is shown in Table 1, the fluorescence quantum yields of pH-A and pH-B are 0.19 and 0.095 in methanol. As far as we know, only a few near-infrared pH probes possessing a neutral pK_a have been reported, and most of them have extremely low fluorescence quantum yields.^12–14 Among them, the highest one is 0.13.²⁰ Therefore, compared with the previous NIR neutral-pH probes, the quantum yields of pH-A and pH-B are considerably higher.

Table 1 Photophysical properties of pH-A and pH-B

			pH-A	pH-B	C
a Reported 3,5-bi(p-methoxy)phenyl-1,7-bi(p-bromo)phenyl aza-BODIPY (Φf 0.42, in toluene) is used as the standard.
CH3OH	λabs (nm)		703	721	697
	ε (M^−1 cm^−1)		93 000	30 000	11 0000
	λem (nm)		728	738	728
Φf	CH3OH		0.2^a	0.095	0.41
	CH3OH : PBS = 1 : 5	pH = 4	0.08	0.04	—
		pH = 5	0.09	0.06	—
		pH = 6	0.1	0.03	—
		pH = 7	0.1	—	—
		pH = 8	—	—	—

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We have synthesized new near-infrared fluorescent probes, named pH-A and pH-B. Probe pH-A is synthesized from compound 3 and compound 5 in acetic acid (Scheme 1 and S1 in the ESI†). Compound 5 is synthesized according to the literature.^19,21 Probe pH-B is obtained from pH-A which reacts with SOCl₂ in methanol. The dye-C is synthesized according to the literature.¹⁹ Then the fluorescence quantum yields are measured in methanol. The fluorescence quantum yields are 0.19 for pH-A, 0.095 for pH-B, and 0.4 for C (the standard aza-BODIPY).²²

Scheme 1 Synthesis of pH-A, pH-B and dye-C.

Spectroscopic studies of probes pH-A and pH-B in various buffer solutions and different pH values are undertaken using UV/Vis absorption and fluorescence spectroscopy. The probes display sensitive absorption and fluorescence spectroscopic responses to changes in pH values. As shown in Fig. 1a–d, probe pH-A has the maximum absorption band at 713 nm due to the S0 → S1 transition, a shoulder peak at 660 nm, and strong fluorescence emission at 740 nm in pH 6.4 media. Upon excitation at λ = 695 nm, the near-infrared emission intensity at λ = 740 nm of pH-A has a large decrease with the changes in pH values from 6.4 to 8.5. And the fluorescence intensity of pH-A displays linear responses to pH values in the range from 6.8 to 7.8. The analysis of fluorescence intensity of pH-A as a function of pH by using the Henderson–Hasselbach-type mass action equation yields a pK_a value of 7.2. The absorption of pH-A also has a large decrease with the changes in pH from 6.4 to 8.5 (Fig. 1c and d).

Fig. 1 10 mM fluorescent probe at different pH values in buffer solution containing 20% ethanol. (a) Fluorescent spectra of pH-A at different pH values (pH values from 6.4 to 8.5); (b) pH effect on fluorescence intensity of pH-A (pH values from 4.0 to 9.6); (c) absorption spectra of pH-A at different pH (pH values from 4.2 to 9.2); (d) pH effect on absorption of pH-A at 713 nm (pH values from 4.2 to 9.2); (e) fluorescent spectra of pH-B at different pH values (pH values from 5.4 to 7.5); (f) pH effect on fluorescence intensity of pH-B (pH values from 3.8 to 7.9); (g) absorption spectra of pH-B at different pH (pH values from 4.5 to 8.2); and (h) pH effect on absorption of pH-B at 748 nm (pH values from 4.5 to 8.2).

As shown in Fig. 1e–h, probe pH-B has the maximum absorption band at 745 nm due to S0 → S1 transition, a shoulder peak at 690 nm, and strong fluorescence emission at 753 nm in pH 5.0 media. Upon excitation at λ = 705 nm, the near-infrared emission intensity at λ = 753 nm of pH-B shows a large decrease with the changes in pH values from 5.4–7.2. The analysis of fluorescence intensity of probe pH-B as a function of pH by using the Henderson–Hasselbach-type mass action equation yields a pK_a value of 6.1. And there is a minor change of absorption with the increase of pH.

There is a large difference of the changes in the absorption spectra between probe pH-A and probe pH-B (Fig. 1c, d, g, and h), owing to the different effects of the pH to the conjugation skeleton structures. In basic solution, pH-A exists in the form of cyclolactam leuco (Scheme 2) which possesses a short conjugation length and thus a small absorption coefficient in a long wavelength region. With the decrease of pH, the transformation from cyclolactam to the ring-open chromophore takes place, so that long wavelength absorption increases remarkably. For pH-B, it has been esterified, so it cannot form cyclolactam leuco, and exists always in the form of the ring-open chromophoric structure. This is why pH-B shows stable absorption spectra without influence by pH changes.

Scheme 2 The structures of pH-A and pH-B changes with pH.

To determine any interference on pH measurement by biological molecules, we measure the fluorescence spectra using probe pH-A and pH-B in the presence of metal ions (Fig. 2: Na⁺, K⁺, Ca²⁺, Mg²⁺, Cu²⁺, Fe²⁺, Fe³⁺, Al³⁺, Mn²⁺, Ba²⁺, Ag⁺, Cd²⁺, Co²⁺, Hg²⁺, Zn²⁺, Pb²⁺, and Cr²⁺). Fluorescent probe pH-A displays no response to 200 μM Na⁺, K⁺, Ca²⁺, Zn²⁺, Al³⁺, Mn²⁺, and Ba²⁺. But it responds to 200 μM Cu²⁺, Fe²⁺, Fe³⁺, Ag⁺, Cd²⁺, Co²⁺, Hg²⁺, Zn²⁺, Pb²⁺, and Cr²⁺. Fluorescent probe pH-B displays no response to metal ions, such as Na⁺, K⁺, Ca²⁺, Zn²⁺, Cu²⁺, Fe²⁺, Fe³⁺ Al³⁺, and Mn²⁺ (Fig. 2). But it responds to 200 μM Ag⁺, Cd²⁺, Co²⁺, Hg²⁺, Zn²⁺, Pb²⁺, and Cr²⁺.

Fig. 2 (A) Fluorescent responses of 2.5 μM fluorescent probe pH-A to pH at 5.8 in the absence and presence of different metal ions (200 μM); and (B) fluorescent responses of 2.5 μM fluorescent probe pH-B to pH at 4.84 in the absence and presence of different metal ions (200 μM).

To confirm the applicability of pH-A and pH-B in living cells, we attempt to label MCF-7 cells. As shown in Fig. 3 and 4, the probes are easy to stain in MCF-7 cells and to obtain high resolution fluorescent images. A commercially available mitochondrial dye (Rh123) and a commercially available lysosome dye (DND 189) are employed for the colocalization study. As shown in Fig. 3, we can draw the results that pH-A merges well with fluorescence images of the lysosome dye (Pearson coefficient 0.87, Fig. 3F) and that pH-B merges very well with the fluorescence images of the lysosome dye (Pearson coefficient 0.95, Fig. 3L).

Fig. 3 Colocalization imaging studies of pH-A and pH-B in MCF-7 cells. (A and G) Rh123 Channel 1: λ_ex = 488 nm, λ_em = 498–560 nm. (B and E) pH-A Channel 2: λ_ex = 635 nm, λ_em = 655–755 nm. (D and J) DND 189 Channel 1: λ_ex = 488 nm, λ_em = 498–560 nm. (H and K) pH-B Channel 2: λ_ex = 635 nm, λ_em = 655–755 nm. (C) Overlay of (A and B) (Pearson coefficient 0.67). (F) overlay of (D and E) (Pearson coefficient 0.87). (I) overlay of (G) and (H) (Pearson coefficient 0.69). (L) overlay of (J and K) (Pearson coefficient 0.95).

Fig. 4 Fluorescence images of MCF-7 cells incubated with fluorescent probes pH-A and pH-B. (A) Cells are incubated with 8 μM pH-A for 15 min. (B) Cells are incubated with 8 μM pH-A for 15 min and then treated with 1.6 mM chloroquine for 50 min. (C) Cells are incubated with 1.6 mM chloroquine for 30 min and then treated with 8 μM pH-A for 15 min. (D) Cells are incubated with 10 μM pH-B for 20 min. (E) Cells are incubated with 10 μM pH-B for 20 min and then treated with 1.6 mM chloroquine for 50 min. (F) Cells are incubated with 1.6 mM chloroquine for 30 min and then treated with 10 μM pH-B for 20 min. λ_ex = 635 nm, λ_em = 655–755 nm.

To evaluate the probe performance of pH, chloroquine is used for MCF-7 cells. Chloroquine is an alkaline drug which can concentrate lysosomes and raise the pH of the lysosomes simultaneously.^23,24 We divide the imaging experiments into three sets. The first one is the control experiment, in which cells are incubated with probes without adding chloroquine (Fig. 4A and D). In the second set, the cells are stained with the probes and then are treated with chloroquine (Fig. 4B and E). In the third set, chloroquine addition and treatment are performed before cell labelling with the probes (Fig. 4C and F).

For pH-A, the intracellular fluorescence signals are always strong, no matter adding chloroquine or not, and no matter the order of chloroquine treatment and pH-A staining. There is little difference of fluorescence intensity of pH-A between the cells in Fig. 4A–C. As is known, lysosomes are acidic (pH 4–6). Chloroquine can lift the lysosomal pH to some extent, but the final pH is still in the acidic range. Hence, the fluorescence of pH-A, with a higher pK_a value (7.2) closed to neutral pH, is slightly affected by chloroquine. In contrast, for pH-B, there is a very large difference of fluorescence intensity between the cells in Fig. 4D–F. This difference can be ascribed to the acidic pK_a of pH-B at 6.1. Compared with Fig. 4D (control), Fig. 4E and F demonstrated remarkably lower fluorescence intensities. Moreover, fluorescence in Fig. 4F is too weak to be detectable, which is even weaker than Fig. 4E. This phenomenon indicates that pre-treatment of chloroquine could reduce the amount of pH-B accumulated in the lysosomes. Therefore, lysosomal pH increased by chloroquine not only quenches the fluorescence of pH-B but also lowers the cell uptake of pH-B. And based on the above results of the two pH probes, we can draw a conclusion that the probes can monitor changes of pH in lysosomes.

In order to further prove the applicability of the probes, flow cytometry (FCM) is used to quantitatively evaluate the changes of fluorescence intensity with pH. Kim’s group, Ohkuma’s group and Baggaley’ group report that the intracellular pH changes with extracellular pH.^11,22,25 MCF-7 cells are incubated with the probes at pH = 4.5, 5.5, 6.5, 7.5, and 8.6 and then they are used for FCM. The changes of intracellular fluorescence intensity indicated by the shift of the fluorescence signal are measured in Fig. 5. The number of the cells with fluorescence intensity increased as the cells are incubated with pH-A at pH from 4.5 to 6.5 (Fig. 5A, a, b, c). The fluorescence intensity increases little because of its pK_a close to neutral. And the fluorescence intensity decreases, as the cells are incubated with pH-A at pH from 6.5 to 8.6 (Fig. 5B, c, d, e). We can know that pH-A is able to monitor the change of pH in the cells. Similar results are obtained from flow cytometry experiments in which cells are incubated with pH-B at pH = 4.5, 5.5, 6.5, 7.5, and 8.6 (Fig. 5C and D). The change in the magnitude of fluorescence intensity of pH-B is greater than that of pH-A via Fig. 5A–D, which also proves the above result, that there is a large difference of fluorescence intensity of pH-B after the cells are incubated with chloroquine. And that also proves that the probes can monitor the changes of pH in the cells.

Fig. 5 Flow cytometric analysis of MCF-7 cells. (A) MCF-7 cells incubated with pH-A (6 μM) at pH = 4.5 (a), 5.5 (b), 6.5 (c). (B) MCF-7 cells incubated with pH-A (6 μM) at pH = 6.5 (c), 7.5 (d), 8.6 (e). (C) MCF-7 cells incubated with pH-B (8 μM) at pH = 4.5 (f), 5.5 (g), 6.5 (h). (D) MCF-7 cells incubated with pH-B (8 μM) at pH = 6.5 (f), 7.5 (g), 8.6 (h).

In summary, we have prepared two near-infrared fluorescent probes for pH (pH-A and pH-B). And the fluorescence quantum yields of pH-A and pH-B are 0.19 and 0.095. As far as we know, it is not reported for pH in the near-infrared range of fluorescent probes that there is a high fluorescence quantum yield (pH-A, Φ_f 0.19 in methanol and pH-B, Φ_f 0.095 in methanol) and its pK_a close to neutral pH. And the applicability of these probes has been confirmed by monitoring the rise of lysosomal pH in MCF-cells stimulated by chloroquine. We believe that these pH probes may find more applications in detecting pH values in vivo and exploring the function of cells.

Acknowledgements

This work is supported by the National Natural Science Foundation of China (No. 2117402221376038, and 21421005 and 21576040), and National Basic Research Program of China (No. 2013CB733702).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Details of the synthesis, NMR spectroscopic, ¹HNMR, ¹³CNMR, cell culture and confocal imaging. See DOI: 10.1039/c6ra11637j

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