Near-Infrared Probe Based on Rhodamine Derivative for Highly

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Near-infrared probe based on rhodamine derivative for highly sensitive and selective lysosomal pH tracking Guangle Niu, Panpan Zhang, Weimin Liu, Mengqi Wang, Hongyan Zhang, Jiasheng Wu, Li-Ping Zhang, and Pengfei Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04417 • Publication Date (Web): 04 Jan 2017 Downloaded from http://pubs.acs.org on January 5, 2017

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Analytical Chemistry

Near-infrared probe based on rhodamine derivative for highly sensitive and selective lysosomal pH tracking Guangle Niu,†,‡,⊥ Panpan Zhang,†,§,⊥Weimin Liu,*,†,‡ Mengqi Wang,† Hongyan Zhang,† Jiasheng Wu,† Liping Zhang,†,‡ and Pengfei Wang†,‡ †

Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of

Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190, China ‡

School of Future Technology, University of Chinese Academy of Sciences, Beijing, 100049, China

§

Institute of Functional Nano and Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based

Functional Materials and Devices, Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Suzhou 215123, China ⊥

G.N. and P.Z. contributed equally

*[email protected]

TOC

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ABSTRACT

The development of near-infrared fluorescent probes with low pKa, high selectivity, high photostability, and high sensitivity for lysosomal pH detection is of great importance. In the present work, we developed a novel near-infrared lysosomal pH probe (Lyso-hNR) based on a rhodamine derivative. Lyso-hNR showed fast, highly sensitive, and highly selective fluorescence response to acidic pH caused by the H+-induced structure changes from the non-fluorescent spirolactam form to the highly emissive open-ring form. Lyso-hNR displays a significant fluorescence enhancement at 650 nm (over 280-fold) from pH 7.0 to 4.0 with a pKa value of 5.04. Live cell imaging data revealed that Lyso-hNR can selectively monitor lysosomal pH changes with excellent photostability and low cytotoxicity. In addition, Lyso-hNR can be successfully used in tracking lysosomal pH changes induced by chloroquine and those during apoptosis. All these features render Lyso-hNR a promising candidate to investigate lysosome-associated physiological and pathological processes.

INTRODUCTION

As important acidic organelles in eukaryotic cells, lysosomes (pH 4.5−5.5) play key roles in numerous cellular biological processes,1–3 such as endocytosis,4 cell growth and apoptosis,5,6 autophagy,7 ion metabolism,8 and oxidative stress.9 Aberrant fluctuation of lysosomal pH causes lysosome malfunction, which leads to several lysosomal storage diseases, cancer, and Alzheimer’s disease.10–12 Therefore, effective techniques to investigate real-time lysosomal pH changes are of significant importance to study lysosome-associated physiological and pathological processes.13–16

Fluorescent detection has become an indispensable tool to investigate biological events because of its simple operation, fast response, high selectivity, high sensitivity, and unparalleled spatiotemporal ACS Paragon Plus Environment

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resolution.17–21 Of particular interest is developing long-wavelength emissive probe especially in the near-infrared (NIR) region (650–900 nm) because of minimum photodamage to biological samples, deep tissue penetration, and minimum interference from background autofluorescence in living systems.22–25 Fluorescent probes have been developed for lysosomal pH detection.26–43 However, the development of NIR lysosome-targeting pH probes is very difficult because of its special requirements, such as high OFF–ON emission ratio, lack of background fluorescence under neutral conditions, and suitable pKa. Although several NIR fluorescent probes have been developed for lysosomal pH detection,44–49 there are some limitations including low fluorescence quantum yield at acid media, high pKa values (> 6.0), interference by reactive oxygen species such as H2O2, and high background fluorescence in live samples. Therefore, the development of novel NIR lysosomal pH probes with suitable pKa, high selectivity, high photostability, and high sensitivity is of great importance. Rhodamine dyes have attracted much interest in biomolecular detection and biomedical imaging for their outstanding photophysical and chemical properties.50–52 Significantly, the strategy, originally proposed by Czarnik’s group,53 has been widely utilized as an effective platform for a large number of targets54–56 in biological systems by turning the non-fluorescent spirocyclic form into the highly fluorescent open-ring form.57,58 More importantly, the non-fluorescent spirolactam forms of rhodamine-based probes can be tuned by H+-induced ring-opening, thereby causing significant fluorescence enhancement.59,60 Some lysosome-targeting fluorimetric pH probes based on rhodamine dyes have been developed.27,28,30–32,35–41 However, the short absorption and emission (mainly in visible region) of almost all the rhodamine derivative-based lysosomal pH probes restrict their applications in living systems.46,48 Wang and co-workers synthesized a NIR probe (SiR-B) based on the spiroboronate Si-rhodamine for lysosomal pH imaging. However, the probe SiR-B had a high pKa = 6.2 and H2O2 could quench its fluorescence.48 ACS Paragon Plus Environment


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Scheme 1. Molecular structures of H-hNR, hNR, and Lyso-hNR. Our group recently synthesized a novel deep-red emissive xanthene derivative (H-hNR, Scheme 1) with high fluorescence quantum yield (0.31 in Tris−HCl−EDTA buffer) for rapid live cell imaging.61 The hybrid of H-hNR and benzoic acid could lead to new highly-emissive rhodamine derivative with deep-red to near-infrared emission (hNR, Scheme 1).62 We anticipated that novel spirolactam-based probes can be designed by the modification of the benzoic acid group in hNR. Herein, we synthesized a novel lysosomal pH probe (Lyso-hNR) via chemical conjunction of hNR and the lysosome-targeting group 4-(2-aminoethyl)morpholine (Scheme 1). Lyso-hNR showed NIR fluorescence (λem = 650 nm) by responding to acidic pH. The detailed pH titration, reversibility, and selectivity of the probe were systemically investigated. Furthermore, the potential applications of Lyso-hNR in live samples were further evaluated via fluorescence imaging techniques.

EXPERIMENTAL SECTION Materials and Methods. All commercial chemicals were purchased from commercial suppliers and used without further purification. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium (MTT), 4-(2-aminoethyl)morpholine, N-hydroxysuccinimide (NHS), and N,N´-dicyclohexylcarbodiimide (DCC) were purchased from Sigma-Aldrich. Nigericin, monensin, and dexamethasone were purchased from J&K Chemical Co., Ltd. Chloroquine diphosphate was purchased from Tokyo Chemical Industry Co., Ltd. LysoTracker Green DND-26 (LTG) and LysoTracker Deep Red (LTDR) were purchased from Invitrogen ACS Paragon Plus Environment

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(USA). Concentrated H2SO4 and HClO4 (70%) were purchased from Beijing Chemical Regent Co., Ltd. Anhydrous CH2Cl2 was distilled by refluxing in presence of CaH2. The Britton–Robinson (BR) buffers were prepared by mixing 40 mM acetic acid, boric acid, and phosphoric acid. The pH was adjusted by different amounts of diluted NaOH or HCl solutions. 1H NMR (400 MHz) and

13

C NMR (100 MHz)

spectra were obtained on a Bruker Advance-400 spectrometer, with tetramethylsilane as an internal standard. The HRMS spectra were measured with Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS). All absorption and fluorescence spectra in this work were obtained with Hitachi U3010 and Hitachi F4500 fluorescence spectrometers at room temperature, respectively. The fluorescence quantum yield of Lyso-hNR in BR buffer (40 mM, pH = 4.0) was determined by using cresyl violet in methanol (Φ = 0.54) as the standard.63 Imaging. Two tumor cell lines, A549 (human lung adenocarcinoma epithelial cell line) and HeLa (human cervical adenocarcinoma epithelial cell line), were cultured in confocal dishes in culture media (McCoy's 5A and DMEM, respectively, which was supplemented with 10% FBS, 50 U/mL penicillin, and 50 mg/mL of streptomycin) under 5% CO2/air at 37 °C in a humidified incubator. The cells were incubated for 24 h prior to the imaging experiments. Fresh culture media (containing 2.5 µM Lyso-hNR and 100 nM LTG for co-localization experiments) were added and further incubated at 37 °C under 5% CO2 for 25 min before imaging. Cells were washed with phosphate-buffered saline (PBS, pH = 7.4). Confocal images were acquired with a Nikon C1si laser scanning confocal microscopy (for LTG, excited at 488 nm, emission was collected in the FITC channel; for Lyso-hNR, excited at 640 nm, emission was collected in the Cy5 channel) equipped with a 60× oil immersion objective lens. Images were processed by the NIS-Elements Viewer 3.20. To estimate the cellular pH in live cells with the probe Lyso-hNR, a previously published method was followed.42



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Synthesis of hNR. The compound hNR was synthesized by following our previous method.64 Compound 165 (189 mg, 1 mmol) and compound 2 (313 mg, 1 mmol) were successively added to the stirred solution of concentrated H2SO4 (10 mL) in a round-bottomed flask. After heating at 100 °C for 4 h. The mixture was cooled to room temperature and poured into ice water. HClO4 (1 mL) was slowly added to the solution; the resulting precipitate was filtered and washed with cold water. The sample was dried under vacuum condition and purified by silica gel chromatography with v(CH2Cl2)/v(CH3OH) (from 20:1, 10:1 to 8:1) as the eluent to yield hNR as a green solid (490 mg, 86%). 1H NMR (400 MHz, DMSO-d6) δ ppm 13.20 (s, 1H), 8.20 (s, 1H), 8.18 (d, J = 2.6 Hz, 1H), 7.86 (t, J = 7.5 Hz, 1H), 7.75 (t, J = 7.6 Hz, 1H), 7.41 (d, J = 7.5 Hz, 1H), 7.24 (d, J = 2.1 Hz, 1H), 7.09 (dd, J = 9.4, 2.2 Hz, 1H), 6.93 (dd, J = 9.2, 2.3 Hz, 1H), 6.87 (d, J = 9.4 Hz, 1H), 6.75 (d, J = 1.9 Hz, 1H), 3.58 (q, J = 7.0 Hz, 4H), 3.19 (s, 6H), 2.97 – 2.78 (m, 2H), 2.55 – 2.37 (overlapped with DMSO-d6, m, 2H), 1.20 (t, J = 7.0 Hz, 6H). 13C NMR (100 MHz, DMSO-d6): δ ppm 166.5, 163.3, 156.1, 155.0, 153.0, 145.0, 134.5, 133.0, 130.8, 129.9, 129.2, 129.0, 128.6, 118.3, 115.2, 114.2, 113.0, 112.0, 110.7, 96.0, 40.0 (overlapped with DMSO-d6), 44.8, 26.9, 23.5, 12.4. HRMS-ESI m/z calcd for [C30H31N2O3]+ 467.23292, found 467.23312. Synthesis of Lyso-hNR. The solution of hNR (283 mg, 0.5 mmol) in anhydrous CH2Cl2 (20 mL) was successively added with NHS (92 mg, 0.8 mmol) and DCC (103 mg, 0.5 mmol). The mixture was stirred at room temperature for 0.5 h. A solution of anhydrous CH2Cl2 (10 mL) containing 4-(2-aminoethyl)morpholine (390 mg, 3 mmol) and NEt3 (303 mg, 3 mmol) was added dropwise to the solution, which was stirred overnight. The mixture was filtered to remove insoluble solids, and the filtrate was extracted with CH2Cl2, washed with saturated brine, and dried over anhydrous MgSO4. The solvent was removed under reduced pressure, and the residues were purified by silica gel chromatography with v(CH2Cl2)/v(CH3OH) (from 60:1, 40:1 to 20:1) as the eluent to yield Lyso-hNR as a white solid (63 mg, 22%).

1

H NMR (400 MHz, CDCl3) δ ppm 7.89 – 7.84 (m, 1H), 7.71 (d, J = 8.6 Hz, ACS Paragon Plus Environment

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1H), 7.49 – 7.41 (m, 2H), 7.18 (dd, J = 5.7, 2.2 Hz, 1H), 6.64 (dd, J = 8.6, 2.6 Hz, 1H), 6.49 (d, J = 2.3 Hz, 1H), 6.42 (d, J = 2.5 Hz, 1H), 6.38 (d, J = 8.8 Hz, 1H), 6.27 (dd, J = 8.9, 2.5 Hz, 1H), 3.66 – 3.45 (m, 4H), 3.34 (q, J = 7.0 Hz, 4H), 2.99 (s, 6H), 2.73 – 2.55 (m, 2H), 2.12 – 2.53 (m, 6H), 1.84 – 1.55 (m, 4H), 1.17 (t, J = 7.0 Hz, 6H).

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C NMR (100 MHz, DMSO-d6): δ ppm 167.0, 152.3, 150.9, 150.6, 148.2, 146.4,

137.3, 132.5, 131.5, 128.5, 128.4, 123.3, 122.8, 122.2, 117.2, 111.2, 109.5, 108.6, 104.8, 100.0, 97.4, 66.0, 65.4, 56.0, 53.1, 43.6, 36.5, 28.0, 20.9, 18.5, 12.4. HRMS-ESI m/z calcd for [C36H43N4O3]+ 579.33297, found 579.33320.

RESULTS AND DISCUSSION

Synthesis of Lyso-hNR. The lysosomal pH probe Lyso-hNR was readily synthesized in two steps as illustrated in Scheme 2. Compound 1 was synthesized and reported by our previous work.65 Condensation of compounds 1 and 2 in heated and concentrated H2SO4 produced hNR.64 Lyso-hNR was obtained from hNR via a typical amide reaction with 4-(2-aminoethyl)morpholine in anhydrous CH2Cl2. The final structure of Lyso-hNR was characterized by 1H NMR, 13C NMR, and HRMS.

O O

+ N

N

1

OH

COOH

COOH concentrated H2SO4 100°C then HClO4

ClO4N

2

O N

hNR O

O

N N

hNR

+

NHS, DCC, NEt3

H2N N

O anhydrous CH2Cl2

N

O

Lyso-hNR

N

Scheme 2. Synthesis of lysosomal pH probe Lyso-hNR.

Spectroscopic properties of Lyso-hNR to pH. The spectroscopic properties of Lyso-hNR were examined in Britton–Robinson (BR) buffers (40 mM) containing 1% DMSO at different pH; the ACS Paragon Plus Environment


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corresponding spectra are shown in Figure 1. As shown in Figures 1a–b, the solution of Lyso-hNR was colorless and non-fluorescent when the pH of BR buffer was higher than 7.0 due to its stable non-fluorescent spirolactam form. When the pH decreased from 7.0 to 4.0, the solution was blue colored with the appearance and a dramatic increase of an intense band at 610 nm (Figure 1a). Concomitantly, a significantly enhanced NIR fluorescence signal at 650 nm (Figure 1b) appeared because of the H+-induced ring opening of spirolactam (more than 280-fold increase), thereby demonstrating that Lyso-hNR is a very sensitive acid-responsive probe. Notably, the measured fluorescence quantum yield of Lyso-hNR in acidic condition (pH = 4.0) was 0.24. The pH titration curve showed that the changes in the fluorescence intensity (at 650 nm) as a function of pH yielded a pKa of 5.04 (Figure 1c), thereby indicating that Lyso-hNR could detect lysosomal pH changes in live cells. Lyso-hNR also displayed good linearity (R2 = 0.98499) of the fluorescence intensity versus pH values in the range of 4.4–5.6 (Figure S1). The time courses of fluorescence intensity of Lyso-hNR in BR buffers of various pH values (4.0 and 7.0) were further examined, and the fluorescence intensity could reach saturation in approximately 1 min (Figure S2). Such a rapid respond time is vital for Lyso-hNR to track lysosomal pH changes in real time. Moreover, Lyso-hNR had excellent reversibility between pH 4.0 and 7.0 (Figure 1d).


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Figure 1. (a) Absorption and (b) fluorescence spectra of Lyso-hNR (10 µM) at different pH values in BR buffer solution. Inserts in (a) and (b) show the photographed and fluorescence images of the Lyso-hNR solution under UV light (365 nm), respectively. (c) The pH titration curve was plotted by NIR fluorescence as a function of pH. (d) pH reversibility study of Lyso-hNR between pH 7.0 and 4.0 in BR buffer solution. Conditions: λex = 580 nm, λem = 650 nm. Specific response to acidic pH. The selectivity of probe Lyso-hNR for preferential binding to protons over other potential interferents under biological conditions was investigated by fluorescence spectroscopy in the BR buffer solution at different pH. As shown in Figure 2, the fluorescence intensity of Lyso-hNR at pH = 7.0 showed negligible changes upon addition of common cations, such as Na+, K+, Ca2+, and Mg2+, or heavy and transition-metal ions, such as Hg2+, Cd2+, Co2+, Ni2+, Zn2+, Mn2+, Cu2+, Pb2+, Ba2+, and Fe3+. In addition, the fluorescence intensity of Lyso-hNR generally remained stable in the presence of common anions (Ac-, HS-, H2PO4-, HPO42-, and CO32-). Various biological molecules, including amino acids, glucose, and reactive oxygen species (HClO or H2O2), exhibited negligible interference on the ACS Paragon Plus Environment


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fluorescence of Lyso-hNR. Meanwhile, the aforementioned analytes had no influence on the fluorescence intensity of Lyso-hNR at pH = 4.0. These results demonstrated that Lyso-hNR exhibits specific fluorescent response to acidic pH with negligible interference from other analytes.

Figure 2. Fluorescence responses of the probe Lyso-hNR (10 µM) to different potential interferents in pH 4.0 and 7.0 BR buffer solutions: (1) control; (2) Na+; (3) K+; (4) Ca2+; (5) Mg2+; (6) Hg2+; (7) Cd2+; (8) Co2+; (9) Ni2+; (10) Zn2+; (11) Mn2+; (12) Cu2+; (13) Pb2+; (14) Ba2+; (15) Fe3+; (16) Ac-; (17) HS-; (18) H2PO4-; (19) HPO42-; (20) CO32-; (21) Cys; (22) Hcy; (23) GSH; (24) Ser; (25) Arg; (26) Lys; (27) Thr; (28) Leu; (29) Val; (30) Trp; (31) Glu; (32) His; (33) Asp; (34) Tyr; (35) glucose; (36) HClO; (37) H2O2. Concentration: 0.2 mM for (6)–(15); 1 mM for others. Conditions: λex = 580 nm, λem = 650 nm. Fluorescence imaging in living samples. To further evaluate the potential biological applications of the probe Lyso-hNR in living samples, cell imaging experiments were performed in A549 and HeLa cells by confocal laser scanning microscopy (CLSM). We first performed imaging experiments in A549 cells at different time-points. As seen in Figures 3 and S3, the fluorescence intensity in the cytoplasm almost reached saturation in approximately 25 min. Bright spots with NIR fluorescence could be observed in cytoplasm near the perinuclear regions of live A549 cells (Figure 3), which were also observed in live ACS Paragon Plus Environment

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HeLa cells (Figure S4). Imaging experiments in HeLa cells stained with different concentrations of Lyso-hNR were further performed. After incubation for 25 min, the clear NIR fluorescence of Lyso-hNR was detected at concentrations as low as 1 µM (Figure S5) because of its high fluorescence quantum yield in acidic media.

Figure 3. CLSM images of A549 cells stained with Lyso-hNR (3 µM) at different time-points. Scale bar: 10 µm. 4-(2-Aminoethyl)morpholine is a lysosome-targeting functional group;66–68 thus, we anticipated that Lyso-hNR could selectively stain lysosomes in live cells. Consequently, colocalization experiments with the commercial lysosome dye LysoTracker Green DND-26 (LTG) were conducted to identify the intracellular location of Lyso-hNR. Lyso-hNR and LTG had nearly the same distributions in live HeLa cells (Figure 4). The corresponding Pearson’s coefficient and Mander’s overlap coefficient for HeLa cells were 0.92 and 0.93, respectively. In addition, colocalization imaging of A549 cells confirmed that Lyso-hNR had excellent overlap with LTG (Figure S6). The above-mentioned imaging data demonstrated that Lyso-hNR is selectively located in lysosomes in live cells.



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Figure 4. CLSM images of HeLa cells stained with Lyso-hNR (2.5 µM) and LTG (100 nM). Scale bar: 10 µm. (a) Fluorescence image of Lyso-hNR in the Cy5 channel. (b) Fluorescence image of LTG in the FITC channel. (c) Bright-field image. (d) Overlay of (a), (b), and (c). (e) Intensity profile within the regions of interest (yellow line in Figures 4a–d) of Lyso-hNR and LTG across HeLa cells. (f) The fluorescence intensity correlation plot of Lyso-hNR (Cy5 channel) and LTG (FITC channel). Then we tested the ability of Lyso-hNR to estimate cellular pH values in live cells. HeLa cells were incubated with Lyso-hNR (2.5 µM) for 30 min; the media were replaced with various PBS buffers containing 10 µM nigericin and 5 µM monensin at different pH (pH 4.0, 4.5, 5.0, 5.5, 6.0, and 7.0). After incubation for another 30 min, the confocal dishes were washed with the corresponding PBS buffers at the respective pH values before cell imaging was performed. The NIR fluorescence intensity of Lyso-hNR in HeLa cells was decreased from pH = 4.0 to 7.0 because of the formation of non-fluorescent spirolactam (Figures 5 and S7). Almost no fluorescence could be detected in HeLa cells at pH value 7.0. It should be noted that NIR fluorescence of Lyso-hNR was distributed throughout the whole cell, and this abnormal phenomenon was also observed in literature34,42,69,70. The plot of the qualified mean fluorescence intensities of Lyso-hNR in HeLa cells as a function of pH indicated a pKa value of approximately 4.95


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(Figure S7), which was slightly lower than that obtained in BR buffers (pKa = 5.04; Figure 1c). These data suggested that Lyso-hNR could display good sensitivity in monitoring lysosomal pH changes in live cells.

Figure 5. CLSM images of HeLa cells stained with Lyso-hNR (2.5 µM) in different pH PBS buffers.

Additionally, we studied whether Lyso-hNR could track lysosomal pH changes after treatment with the antimalarial drug chloroquine. After incubation for 25 min, the strong fluorescence of Lyso-hNR was observed in HeLa cells without the addition of chloroquine (Figure 6). However, after treatment with chloroquine (100 µM) for another 10 min, the fluorescence of Lyso-hNR in HeLa cells was significantly reduced (Figure 6), thereby indicating that the lysosomal pH increased probably due to the proton leakage. The leakage of protons at certain lysosomal pH is caused by chloroquine and the formation of non-fluorescent spirolactam.28 Therefore, Lyso-hNR could monitor lysosomal pH changes in real time.



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Figure 6. CLSM images of Lyso-hNR (2.5 µM) in HeLa cells simulated with chloroquine (100 µM). Scale bar: 10 µm. MFI: mean fluorescence intensity.

Lysosomal pH shows obvious increases during cell apoptosis, probably because of lysosomal proton release.71 To further verify the ability of Lyso-hNR to track lysosomal pH, the anti-inflammatory drug dexamethasone was used to induce cell apoptosis. After incubation of Lyso-hNR (2.5 µM) in HeLa cells for 25 min, the cells were washed with PBS twice before dexamethasone (2 µM) was added to the medium. The fluorescence of Lyso-hNR was obtained at different time-points via CLSM (Figures 7 and S8). After the addition of dexamethasone, the fluorescence intensity of Lyso-hNR dramatically decreased in the first 10 min, then slowly decreased as time goes on (Figure S8). This time-dependent decrease in the fluorescence intensity was attributed to apoptosis-induced increase of lysosomal pH, thereby forming the non-fluorescent spirolactam. Apoptosis was also proved by dramatic morphological changes of the cells (Figure 7). This observation is consistent with previously reported data28,36,72 and further demonstrates that Lyso-hNR can track lysosomal pH changes in real time.


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Figure 7. CLSM images of Lyso-hNR (2.5 µM) in HeLa cells undergoing apoptotic death induced by dexamethasone (2 µM) at different time-points. Scale bar: 10 µm.

Cytotoxicity and photostability. Furthermore, we performed standard MTT assays to evaluate the cytotoxicity of Lyso-hNR in live cells. After incubation for 24 h, the cell viabilities of A549 and HeLa cells were extremely high. Even when the concentration of Lyso-hNR reached 10 µM, the cell viabilities were still higher than 85% (Figure 8a). Therefore, Lyso-hNR exhibits very low cytotoxicity. In addition, the photostability of Lyso-hNR was evaluated in HeLa cells by continuous irradiation with a 640-nm laser. After 25 min of irradiation, over 80% of the initial fluorescence intensity still remained (Figure 8b). By comparison, the fluorescence intensities of LTDR and LTG decreased to 51% and 34% of the initial fluorescence intensity, respectively (Figure 8b). The photostability experiment confirmed that Lyso-hNR exhibits high resistance to photobleaching and could be utilized for long-term monitoring the dynamic changes of lysosomal pH in live cells.



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Figure 8. (a) Cytotoxicity of Lyso-hNR in A549 and HeLa cells. (b) Photostability of Lyso-hNR, LTDR, and LTG in HeLa cells under continuous irradiation. Irradiation conditions: for Lyso-hNR and LTDR, 640 nm laser, laser power of 20%; for LTG, 488 nm laser, laser power of 10%.

CONCLUSIONS

A novel NIR probe (Lyso-hNR) was successfully synthesized based on the rhodamine derivative hNR for tracking lysosomal pH in live cells. Lyso-hNR showed NIR fluorescence (650 nm) and remarkable fluorescence enhancement (more than 280-fold) from pH 7.0 to 4.0 because of the H+-induced ring opening of non-fluorescent spirolactam. Notably, Lyso-hNR exhibits a rapid response, suitable pKa (5.04), high sensitivity, high selectivity, good reversibility, excellent photostability, and low cytotoxicity. Lyso-hNR has been successfully used for fluorescent lysosomal pH detection in live cells and tracking


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lysosomal pH changes induced by chloroquine or during apoptosis. The present work provides a facile platform to develop novel rhodamine-based probes for detecting and imaging a wide variety of analytes in living samples. Further work to construct a ratiometric probe based on hNR for precisely mapping lysosomal pH in live cells is of particular importance and will be underway in our laboratory.

ASSOCIATED CONTENT

Supporting Information

Supporting Information Available: Photophysical data; cell data; NMR spectra; HR-MS spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected]. Tel.: +86 10 82543475. ORCID Guangle Niu: 0000-0002-5403-6880 Weimin Liu: Author Contributions ⊥

G.N. and P.Z. contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS



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This work was supported by the NNSF of China (Grant Nos. 21373250 and 21673265) and 973 Program (No. 2014CB932600).

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