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DOI: 10.1039/C9NJ02771H (Paper) New J. Chem., 2019, 43, 13363-13370

Ratiometric emission NIR-fluorescent probe for the detection of lysosomal pH in living cells and in vivo†

Weifen Niu‡ *, Juan Jia‡ , Junkai Li , Chao Zhang and Keming Yun *
School of Forensic Medicine, Shanxi Medical University, Taiyuan 030001, P. R. China. E-mail: niuweifen_2000@163.com

Received 28th May 2019 , Accepted 15th July 2019

First published on 31st July 2019

Abstract

The lysosome is known to be an acidic organelle that is ubiquitous in cells. Acidic pH is a characteristic feature of lysosomes, and aberrant lysosomal pH values have been manifested to be associated with diverse diseases. In order to monitor the lysosomal pH changes, we developed a ratiometric emission NIR-fluorescent probe (CzQl) for the quantitative analysis of the lysosomal pH in living cells and in vivo. CzQl was constructed via the ethylene bridging of carbazole derivatives and quinoline. The protonation of the probe resulted in a marked green-to-red emission color change. The probe exhibited an ideal pKa value of 4.60 with a linear relationship in the pH range of 3.9–5.3, which covered most of the pH ranges of lysosomes, and was useful for the quantitative detection and imaging of lysosomal pH changes. Moreover, CzQl exhibited easy loading capability, high selectivity, a large Stokes shift, specific lysosome-targeting ability and low cytotoxicity; thereby, it was successfully used for monitoring the lysosomal pH changes at a cellular level as well as the LPS-mediated inflammation in vivo.

Introduction

Lysosomes, single-membrane organelles defined by a uniquely strong acidic lumenal pH (4.5–5.5) and high content of acid hydrolases, are the shared degradative compartments of the endocytic and autophagic pathways.1,2 A variety of hydrolases are responsible for substrate degradation.1,3 Besides, lysosomes are the points of particular vulnerability in many neurodegenerative diseases.1,4,5 Beyond their role in substrate degradation, new findings have ascribed to lysosomes a leading role in sensing and responding to cellular nutrients, growth factors and cellular stress.1 However, the acidic pH environment is the crucial biomarker for lysosomes; abnormal fluctuations in the lysosomal pH value can cause lysosomal dysfunction, which is associated with several diseases.5–10 Consequently, the detection of the lysosomal pH value in living cells and in vivo is vital for investigating lysosome-associated physiological and pathological processes.

Organelle-specific fluorescent probes are robust molecular tools for sensing and monitoring trace amounts of biological molecules and the processes of individual subcellular organelles in living cells and in vivo by virtue of their excellent sensitivity and high spatiotemporal resolution. To date, numerous lysosome-selective pH probes have been developed for biological applications.11 Some recent studies on ratiometric fluorescent probes for lysosomal pH sensing published in the last three years have been summarized in Table 1.12–21 Compared with the single emission detection, the emission ratio simultaneously reflects the population of the protonated and deprotonated forms of the fluorescent probe, which can cancel out the most possible effects of environmental variations, probe distribution, and instrumental performance, thus offering more accurate analysis because of its self-referencing capability by the calculation of the two emission intensity ratio.22–25 In addition, as seen in Table 1, some probes have short emission wavelengths, which can be due to the interference from cellular autofluorescence. NIR-fluorescence emissions can largely eliminate the interference of cellular autofluorescence and also reduce light-induced damage to living cells and organisms.26 Moreover, some probes have complex structures and synthesis processes. Thus, the design and development of ratiometric emission NIR-fluorescence probes with simple structures and easy synthesis for sensing the lysosomal pH are of great concern and significantly challenging.

Table 1 Comparison of the properties of recently reported probes for lysosomal pH sensing with the ratiometric method
Probe λ ex/nm λ em/nm pKa Linearity pH range Ref.
a Not reported in the corresponding paper.
image file: c9nj02771h-u1.tif 473 522/557 5.96 3.5–6.0 12
image file: c9nj02771h-u2.tif 375 662/460 5.35 —a 13
image file: c9nj02771h-u3.tif 405 641/483 4.8 — 14
image file: c9nj02771h-u4.tif 470 633/518 3.4 — 15
image file: c9nj02771h-u5.tif 360 514/454 4.46 3.82–5.00 16
image file: c9nj02771h-u6.tif 470 515/665 2.00 — 17
image file: c9nj02771h-u7.tif 495 697/618 4.5 3.5–5.2 18
image file: c9nj02771h-u8.tif 470 613/560 5.00 4.0–6.0 19
image file: c9nj02771h-u9.tif 385 540/425 5.02 3.0–5.0 20
image file: c9nj02771h-u10.tif 380 511/439 — 3.8–6.0 21

In this study, a ratiometric emission NIR-fluorescent probe (CzQl) (Scheme 1) was achieved via the chemical conjugation of methylcarbitol-substituted carbazole and quinoline; the methylcarbitol substituent served as the targeting unit for lysosomes, and the quinoline moiety acted as a proton reaction site. Upon protonation, CzQl could exhibit a pronounced pH-dependent red shift in both absorption and emission spectra, which was attributed to the significant enhancement of the electron-withdrawing ability of the protonated quinoline moiety. Remarkably, this probe exhibited an expected pKa value of 4.60 with a linear relationship in the pH range of 3.9–5.3 and could quantitatively detect and image the lysosomal pH changes.


image file: c9nj02771h-s1.tif
Scheme 1 Synthetic scheme and the acid–base form equilibrium of CzQl.

Results and discussion

Optical response to pH

Spectroscopic studies of CzQl in solutions of different pH values were performed using UV-vis absorption spectroscopy and fluorescence spectroscopy. Fig. 1A shows the UV-vis absorption spectral changes of CzQl at various pH values. Under basic-neutral pH conditions, CzQl displayed an absorption maximum (λabs) at 383 nm (εmax = 6.36 × 104 M−1 cm−1). When the pH was changed from neutral (7.0) to acidic (2.5), the absorption maximum shifted to a longer wavelength at 464 nm (εmax = 7.84 × 104 M−1 cm−1) with a well-defined isosbestic point at 415 nm. The red shift in the absorption spectra was attributed to the protonation of the quinoline moiety of CzQl, which enhanced the intramolecular charge transfer (ICT) effect on decreasing pH because of the H+ binding-induced enhancement of the electron-withdrawing ability of quinoline. The 1H NMR titration experiment (Fig. S1, ESI†) showed that the chemical shift values of the quinoline protons H1 and H2 shifted down-field, which confirmed the above conclusion. Furthermore, an easy-to-discern solution color change from nearly colorless to orange was observed on decreasing the pH value (Fig. 1A, inset), which suggested that CzQl could serve as a colorimetric probe in acidic pH conditions.
image file: c9nj02771h-f1.tif
Fig. 1 UV-vis absorption (A) and fluorescence (B) pH titration curves of CzQl (10 μM) with decreasing pH values. (C) Sigmoidal fitting of the pH-dependent emission intensity ratio (F530nm/F637nm). Insets: Photographs of the solution color change (A) and fluorescence change (B) at different pH conditions. (C) The linear relationship over the pH range from 3.9 to 5.3.

The fluorescence spectral changes of CzQl at various pH values are shown in Fig. 1B. When the pH was basic-neutral, the probe exhibited a fluorescence emission maximum at 530 nm (λex = 415 nm, Φ = 0.38) with a large Stokes shift of 115 nm, which could greatly reduce the excitation interference. As the pH decreased from 7.0 to 1.9, the fluorescence intensity at 530 nm was reduced gradually concomitant with the red shift of an emissive band to 637 nm (Δ = 107 nm, Φ = 0.21), leading to an isoemissive point at ∼595 nm. The large red shift in the emission signals was caused by the enhanced ICT effect. Importantly, the marked red shift of the emission spectra offered the possibility to carry out ratiometric detection. With the decrease in pH, the emission ratio of the fluorescence intensities (F530nm/F637nm) also changed dramatically. As seen in Fig. 1C, F530nm/F637nm is reduced by more than 260-fold (from 7.733 to 0.029) over the pH range of 7.0–1.9. The pKa value calculated from the titration curve of the emission ratios (F530nm/F637nm) was 4.60. Interestingly, the plot of F530nm/F637nmversus pH values shows a linear relationship in the pH range of 3.9–5.3, which covers most of the pH ranges of lysosomes. The linear regression equation was F530nm/F637nm = −16.10 + 4.34 pH, with a linear coefficient of 0.9987. Consequently, CzQl showed a marked green-to-red emission color change in response to the change in pH from neutral to acidic (Fig. 1B, inset) and an appropriate pKa value for assessing the change in the lysosomal pH.

It is necessary that CzQl shows a highly selective response towards H+ ions over other potentially competitive species such as various metal ions and some representative bioactive molecules. As shown in Fig. 2, high concentrations (over their physiological concentrations) of metal ions and bioactive molecules cause no significant effect on the fluorescence emission ratio (F530nm/F637nm) of the probe at pH 7.0 and 4.5, respectively. These results confirm that CzQl exhibits an excellent selective response to H+ ions in the presence of metal ions and bioactive molecules.


image file: c9nj02771h-f2.tif
Fig. 2 The fluorescence emission ratios (F530nm/F637nm) of CzQl (10 μM) at pH 7.0 (black bars) and 4.5 (red bars) in the presence of various metal ions and bioactive species. (1) blank; (2) 20 mM K+; (3) 20 mM Na+; (4) 20 mM Ca2+; (5) 20 mM Mg2+; (6) 15 mM Zn2+; (7) 0.15 mM Fe3+; (8) 1 mM Pb2+; (9) 2 mM Cd2+; (10) 5 mM glucose; (11) 5 mM GSH; (12) 2 mM vitamin C; (13) 1 mM leucine; (14) 1 mM valine; (15) 1 mM L-threonine; (16) 1 mM cysteine; (17) 1 mM arginine; (18) 1 mM serine; (19) 1 mM histidine. λex = 415 nm. Data are expressed as mean values with the standard error of the mean of three independent experiments.

Moreover, the emission ratio (F530nm/F637nm) of CzQl was recorded by robustly cycling back and forth between pH 7.0 and 2.2 using concentrated hydrochloric acid and aqueous sodium hydroxide (Fig. S2, ESI†), indicating the reversible sensing ability of CzQl. Also, the response and recovery times in different pH solutions were rapid, i.e., within seconds. Thus, the probe can be used to monitor pH variations in real time. Likewise, the experimental results indicated that the probe solution possessed good photostability (Fig. S3, ESI†). Therefore, this probe shows a promising potential for the quantitative detection of pH changes by means of the ratiometric fluorescence method.

Confocal fluorescence imaging in cells

Because lysosomes are the typical acidic organelles in cells and CzQl has the appropriate pH range for lysosomes, it is practical for the probe to sense this acidity if it can exhibit subcellular localization of lysosomes. To confirm the lysosome-targeting ability of CzQl, colocalization experiments were carried out. B16-F10 cells were stained with CzQl and LysoTracker Red DND-99 (a well-known commercially available lysosome-specific red-emissive probe). In Fig. 3, excellent overlapping images with a high Pearson's co-localization coefficient (A) of 0.93 can be observed (Fig. 3D), clearly demonstrating that CzQl predominantly resided in the lysosomes. Additionally, CzQl exhibited minimal cytotoxicity, which was confirmed by an MTT viability assay (Fig. S4, ESI†).
image file: c9nj02771h-f3.tif
Fig. 3 Colocalization imaging of CzQl and LysoTracker Red DND-99 in B16-F10 cells. (A) CzQl imaging (10 μM, λex= 405 nm; λem= 510–550 nm). (B) LysoTracker Red DND-99 imaging (1.0 μM, λex = 543 nm; λem = 570–610 nm). (C) Overlay of (A) and (B). (D) Intensity correlation plot of CzQl and LysoTracker Red DND-99.

Lysosomal pH imaging

Based on the confirmed intrinsic lysosome-targeting ability, we next tested if CzQl could quantitatively detect the lysosomal pH changes. pH-dependent ratiometric fluorescence imaging of the probe in living cells at different pH conditions was performed; the results are shown in Fig. 4. The excitation wavelength was at 405 nm, and the fluorescence images were recorded at 510–550 nm (green channel) and 620–660 nm (red channel). It was obvious that the cells displayed bright green fluorescence and very weak red fluorescence at pH 7.4. When the extracellular pH decreased to 5.1, the fluorescence in the red channel slightly enhanced. As the pH continued to decrease to 4.0, the green fluorescence intensity was significantly reduced accompanied by the enhancement in red fluorescence. When pH dropped to 3.2, there was faint green fluorescence and strong red fluorescence. The results matched well with those of the fluorescence titrations using the solutions of different pH values. Hence, these outcomes clearly demonstrated that CzQl is capable of monitoring the changes in the lysosome pH values in living cells.
image file: c9nj02771h-f4.tif
Fig. 4 pH-dependent ratiometric fluorescence imaging of CzQl (10 μM) in B16-F10 cells. The excitation wavelength was at 405 nm. The fluorescence images were collected at 510–550 nm (green channel, second column) and 620–660 nm (red channel, third column).

In vivo fluorescence imaging

We next investigated the application of CzQl to visualize the pH changes in vivo induced by lipopolysaccharide (LPS)-triggered inflammation.27,28 As shown in Fig. 5, mouse A is subcutaneously injected with LPS in the peritoneal cavity to cause an acute inflammatory response and then is injected with CzQl. For comparison, mouse B was subcutaneously injected only with CzQl. It was obvious that mouse A (Fig. 5A) treated with both LPS and CzQl exhibited lower fluorescence intensity (pseudo-color) than mouse B treated with only CzQl (Fig. 5B), indicating decrease in the pH value in the inflamed tissues.29–31 As a control, mouse C treated with LPS but not with CzQl showed no fluorescence (Fig. 5C). Therefore, these results demonstrate that CzQl is useful for in vivo imaging and detecting the pH changes in small animals.
image file: c9nj02771h-f5.tif
Fig. 5 In vivo fluorescence images of CzQl in mice with stimulation by LPS. (A) LPS (1.0 mg mL−1 in saline, 200 μL) was injected, followed by injection with CzQl (40 μM, 200 μL). (B) Only CzQl (40 μM, 200 μL) treated. (C) Only LPS (1.0 mg mL−1 in saline, 200 μL) treated. (Ex = 420 nm, Em = 535 nm).

Experimental

Material and apparatus

All chemicals and solvents were of analytical grade and used without further purification. 2-(2-Methoxyethoxy) ethanol was purchased from Aladdin Industrial Inc. Lepidine and p-toluenesulfonyl chloride were purchased from TCI chemical industrial development Co., Ltd. Carbazole was provided by Sigma-Aldrich. Co., Ltd. Trimethyl chlorosilane (TMSCl) and POCl3 were purchased from Alfa Aesar Chemical Co., Ltd. All other commercially available chemicals and solvents were obtained from Beijing Chemical Reagent Co.

1H and 13C NMR spectra were collected in CDCl3 or DMSO-d6 as the solvent with tetramethylsilane (TMS) as the internal standard on a Bruker 400 MHz NMR spectrometer (Bruker biospin, Switzerland). High-resolution mass spectrometry (HRMS) analyses were carried out using a Bruker Autoflex II matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer (Bruker Daltonics, Germany). Absorption spectra were recorded on a Varian Cary 300 UV-vis spectrophotometer (Varian Technology Co., Ltd, USA). Fluorescence spectra were recorded using a PTI Luminescence spectrophotometer. Standard quartz cuvettes with 1 cm light path were used for all optical measurements. Cell imaging was carried out using a confocal laser scanning microscope (Zeiss, LSM880) with a 63× oil-immersion objective lens. In vivo images were obtained using a Bruker small animal in vivo imaging system. Ultrapure water was obtained from a Millipore-Q ultrapurification system. pH measurements were obtained with a Beckman Φ 50 pH meter (Shanghai, China).

Synthesis and characterization of fluorescent probe

Synthesis of 2-(2-methoxyethoxy)ethyl 4-methylbenzenesulfonate (1). A mixture of 2-(2-methoxyethoxy) ethanol (1.20 g, 10 mmol), sodium hydroxide (0.80 g, 20 mmol), THF (20 mL), and water (20 mL) was added into a three-necked flask. The solution mixture was stirred at 0 °C. To the solution mixture, a THF solution (50 mL) of p-toluenesulfonyl chloride (2.28 g, 12 mmol) was added dropwise, and the reaction mixture was stirred for 12 h at room temperature. The reaction mixture was poured into aqueous hydrochloric acid, and the product was extracted with dichloromethane three times. The organic layer was dried over anhydrous magnesium sulfate and filtered and concentrated by evaporation under reduced pressure. The crude product was purified by column chromatography using n-hexane/ethyl acetate (5/1, V/V) as the eluent to give a colorless oil. Yield: 2.38 g (87%).

1H NMR (CDCl3, 400 MHz), δ (ppm): 2.450 (s, 3H), 3.355 (s, 3H), 3.474–3.496 (m, 2H), 3.573–3.596 (m, 2H), 3.682–3.706 (t, J = 4.8 Hz, 2H), 4.160–4.184 (t, J = 4.8 Hz, 2H), 7.332–7.352 (d, J = 8.0 Hz, 2H), 7.793–7.814 (d, J = 8.4 Hz, 2H). 13C NMR (CDCl3, 100 MHz), δ (ppm): 58.91, 67.18, 69.34, 70.59, 71.73, 114.61, 129.72, 131.60, 163.23, 190.41.

Synthesis of 9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole (2). To a solution of carbazole (0.84 g, 5 mmol) in THF (30 mL) at 0 °C, NaH (0.18 g, 7.5 mmol) was added. The solution was stirred for 30 min, and compound 1 (2.06 g, 7.5 mmol) was added with constant stirring. The resulting mixture was stirred overnight at room temperature. The solution was poured into ice water, and the product was extracted with ethyl acetate three times. The organic layer was washed with water and brine and then dried over anhydrous sodium sulfate to remove the solvent. The crude product was separated by silica gel column chromatography with n-hexane and ethyl acetate (3/1) to give the desired product 2 (1.07 g) in 80% yield as a brown oil.

1H NMR (CDCl3, 400 MHz), δ (ppm): 3.310 (s, 3H), 3.405–3.429 (m, 2H), 3.514–3.536 (m, 2H), 3.844–3.876 (t, J = 6.4 Hz, 2H), 4.494–4.526 (t, J = 6.4 Hz, 2H), 7.210–7.262 (m, 2H), 7.420–7.492 (m, 4H), 8.081–8.097 (d, J = 6.4 Hz, 2H). 13C NMR (CDCl3, 100 MHz), δ (ppm): 43.02, 59.01, 69.14, 70.72, 71.79, 108.71, 118.87, 120.20, 122.82, 125.64, 140.51.

Synthesis of 9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole-3-carbaldehyde (3). A 100 mL three-necked flask containing 20 mL dry DMF was cooled to 0 °C and then, POCl3 (0.6 mL, 6 mmol) was added dropwise. The solution mixture was warmed to room temperature and stirred for 1 h. To this reaction mixture, compound 2 (1.07 g, 4 mmol) in dichloromethane (5 mL) was added slowly. The reaction temperature was raised to 75 °C and maintained for 8 h. After cooling to room temperature, the solution was poured into ice water and extracted with dichloromethane. The organic phase was washed with water and brine. Then, the organic layer was dried over anhydrous sodium sulfate, and the solvent was removed. The crude product was purified by silica gel column chromatography using n-hexane/ethyl acetate (3/1) to afford compound 3 (0.82 g) in 69% yield as a yellow solid.

1H NMR (CDCl3, 400 MHz), δ (ppm): 3.276 (s, 3H), 3.375–3.397 (m, 2H), 3.481–3.503 (m, 2H), 3.850–3.879 (t, J = 5.8 Hz, 2H), 4.481–4.510 (t, J = 5.8 Hz, 2H), 7.260–7.329 (m, 1H), 7.468–7.534 (m, 3H), 7.958–7.983 (dd, J = 10.0 Hz, 1.6 Hz, 1H), 8.099–8.118 (d, J = 7.6 Hz, 1H), 8.546–8.549 (d, J = 1.2 Hz, 1H), 10.061 (s, 1H). 13C NMR (CDCl3, 100 MHz), δ (ppm): 43.77, 59.28, 69.49, 71.12, 72.17, 109.64, 109.77, 120.72, 120.90, 123.28, 123.39, 124.05, 126.96, 127.41, 128.95, 141.52, 144.66, 192.05.

Synthesis of 9-(2-(2-methoxyethoxy)ethyl)-3-((E)-2-(quinolin-4-yl)vinyl)-9H-carbazole (CzQl). To the solution of lepidine (0.1 mL, 0.75 mmol) and compound 3 (0.15 g, 0.5 mmol) in DMF (5 mL) in a sealed tube, TMSCl (0.64 mL, 5 mmol) was added, and the resulting mixture was heated to 100 °C for 24 h. After cooling down to room temperature, water was added, followed by aqueous Na2CO3 solution to adjust the pH to 8. The aqueous solution was extracted with dichloromethane three times. The combined organic phase was washed with brine and dried over anhydrous sodium sulfate. After removing the solvent, the crude product was purified by silica gel column chromatography using a dichloromethane and ethyl acetate (5/1) mixture as the eluent to afford CzQl (0.112 g) in 53% yield as an orange solid.

1H NMR (DMSO-d6, 400 MHz), δ (ppm): 3.119 (s, 3H), 3.299–3.323 (t, J = 4.8 Hz, 2H), 3.456–3.480 (t, J = 4.8 Hz, 2H), 3.807–3.834 (t, J = 5.4 Hz, 2H), 4.577–4.604 (t, J = 5.4 Hz, 2H), 7.240–7.277 (t, J = 7.4 Hz, 1H), 7.460–7.495 (t, J = 7.0 Hz, 1H), 7.643–7.701 (m, 3H), 7.780-7.821 (t, J = 8.2 Hz, 2H), 7.890–7.902 (d, J = 4.8 Hz, 1H), 7.948–7.966 (d, J = 7.2 Hz, 1H), 8.032–8.055 (d, J = 9.2 Hz, 1H), 8.111–8.152 (d, J = 16.4 Hz, 1H), 8.225-8.243 (d, J = 7.2 Hz, 1H), 8.614–8.634 (d, J = 8.0 Hz, 1H), 8.670–8.673 (d, J = 1.2 Hz, 1H), 8.876–8.887 (d, J = 4.4 Hz, 1H). 13C NMR (DMSO-d6, 100 MHz), δ (ppm): 54.91, 58.12, 68.88, 69.82, 71.29, 109.45, 109.81, 109.98, 115.96, 118.91, 119.28, 119.62, 120.36, 120.76, 122.60, 122.75, 124.20, 125.02, 125.85, 126.35, 127.68, 129.35, 134.75, 136.15, 140.83, 142.69, 148.47, 150.21. MS (MALDI-TOF) m/z: calcd for [M + H]+, 423.2067; found, 423.2079.

UV-vis and fluorescence pH titrations

A stock solution of CzQl (1 mM) was prepared in DMSO and was further diluted with a mixed solution of DMSO/H2O (1/1, v/v) to an analytical concentration of 10 μM for absorption and emission measurements in quartz cuvettes of 1 cm optical path length (3 mL volume). Slight pH variations in the solution were achieved by adding minimal volumes of HCl (0.01 M). The excitation wavelength was 415 nm. The excitation and emission bandwidths were both set at 1.5 nm. The fluorescence quantum yield was determined using quinine sulfate solution (Φ = 0.577 in 0.1 M H2SO4) at pH 7.0 and fluorescein solution (Φ = 0.95 in 0.1 M NaOH) at pH 1.9 as the reference according to a method described in the literature.32 All spectroscopic experiments were carried out at room temperature.

Colocalization-imaging experiments in B16-F10 cells

B16-F10 cells were cultured in DMEM medium supplemented with 10% FBS and incubated at 37 °C in a 5% CO2 atmosphere and passed and dispersed one day before imaging. Then, the cells were incubated with LysoTracker Red DND-99 (1.0 μM) and CzQl (10 μM) for 30 min. The medium was removed, and the cells were washed with PBS (pH 7.4) three times. Cell imaging was performed using a confocal laser scanning microscope (Zeiss, LSM880) with a 63× oil-immersion objective lens. Confocal images of the cell fluorescence of LysoTracker Red DND-99 were captured using a 543 nm laser, and the collection window was 570-610 nm. The excitation wavelength for CzQl was 405 nm, and the collection window was 510–550 nm.

Intracellular pH imaging

B16-F10 cells were prepared and incubated with 10 μM final concentration of CzQl for 15 min. Then, excess CzQl solution was removed by washing with PBS (pH 7.4) three times, and the cells were incubated with 10 μg mL−1 nigericin in high K+ buffer (including 120 mM KCl, 30 mM NaCl, 0.5 mM MgSO4, 1 mM CaCl2, 20 mM HEPES, 20 mM NaOAc, 1 mM NaH2PO4 and 5 mM glucose) at different pH values (7.40, 5.1, 4.0 and 3.2) for another 15 min. Cell imaging was performed using a confocal laser scanning microscope (Zeiss, LSM880) with a 63× oil-immersion objective lens (λex = 405 nm; λem = 510–550 nm).

In vivo fluorescence imaging

BALB/c mice (20–25 g) were obtained from the Laboratory Animals Center of Shanxi Medical University (Taiyuan, China) for in vivo imaging. All animal experiments were performed in compliance with the Animal Management Rules of the Ministry of Health of the People's Republic of China (Document no. 55, 2001) and approved by the ethics committee of Shanxi Medical University. We have taken great efforts to reduce the number of animals used in these studies; we have also taken efforts to reduce the pain and discomfort experienced by the animals.

According to previous works,33,34 BALB/c mice were given an intraperitoneal injection of LPS (1.0 mg mL−1 in saline, 200 μL). After 4 h, the mice were intraperitoneally injected with CzQl (40 μM, 200 μL) for another 15 min. For comparison, mice with only LPS (1.0 mg mL−1 in saline, 200 μL) treatment for 4 h but no CzQl and with only CzQl (40 μM, 200 μL) treatment for 15 min without stimulation by LPS were prepared. These mice were anesthetized by an intraperitoneal injection of 4% chloral hydrate (300 μL) for 10 min before imaging. In vivo images were then obtained using a Bruker small animal in vivo imaging system with an excitation filter of 420 nm and an emission filter of 535 nm.

Conclusions

In summary, to detect the pH changes in lysosomes, we developed a ratiometric emission NIR-fluorescent probe (CzQl) via an ethylene bridge electron-donating carbazole group π-conjugated with a quinoline electron-accepting moiety, which has an electron donor–acceptor substituted dipolar character. The probe showed red-shifts in its absorption and emission spectra under acidic conditions. Besides, additional advantages were found for CzQl, including a large Stokes shift (>100 nm), good selectivity, high photostability, low cytotoxicity, and excellent cell permeability. All these features enable the probe to successfully achieve the real-time imaging of lysosomal pH in living cells and in vivo.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 21705105). The project was funded by the China Postdoctoral Science Foundation (No. 2019M651087), Scientific and Technologial Innovation Programs of Higher Education Institutions in Shanxi (No. 201802063), and Doctor Fund of Shanxi Province (No. SD1818).

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Footnotes

† Electronic supplementary information (ESI) available: 1H NMR titration, reversibility, photostability cytotoxicity assay, characterization of fluorescent probe. See DOI: 10.1039/c9nj02771h
‡ These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019
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