Hemicyanine-based High Resolution Ratiometric near-Infrared

Jan 20, 2015 - To achieve good adaptability, in this study, a class of resolution-tunable ratiometric NIR fluorescent probes, which possess a stable N...
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Hemicyanine-based High Resolution Ratiometric near-Infrared Fluorescent Probe for Monitoring pH Changes in Vivo Yinhui Li,† Yijun Wang,† Sheng Yang, Yirong Zhao, Lin Yuan, Jing Zheng, and Ronghua Yang* State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China S Supporting Information *

ABSTRACT: Intracellular pH is an important parameter associated with cellular behaviors and pathological conditions. Quantitative sensing pH and monitoring its changes by nearinfrared (NIR) fluorescence imaging with high resolution in living systems are essential but challenging due to the lack of effective probes. To achieve good adaptability, in this study, a class of resolution-tunable ratiometric NIR fluorescent probes, which possess a stable NIR hemicyanine skeleton bearing different substituents, are rationally designed and synthesized, enabling detection through noninvasive optical imaging of organisms. Based on the protonation/deprotonation of the hydroxy group, a marked NIR emission shift provides a ratio signal in response to pH. Meanwhile, two states exhibit good photostability, sensitivity and reversibility, conducive to longtime monitoring of persistent pH changes without disturbance of other biological active species. Among the series, NIR-Ratio-BTZ modified with an electron-withdrawing substituent of benzothiazole exhibited the largest emission shift of about 76 nm from 672 to 748 nm with the pH environment changing from acidic to basic, which could be considered as a good candidate for high resolution pH imaging in live cells, tissues and organisms. Moreover, NIR-Ratio-BTZ has an ideal pKa value (pKa ≈ 7.2) for monitoring the minor fluctuations of physiological pH near neutrality. The ratiometric fluorescence measurement is beneficial to ensure the accuracy of quantitative measuring pH changes, as well as the real-time monitoring pH-related physiological effects both in living cells and living mice. The results demonstrate that NIR-Ratio-BTZ is a highly sensitive ratiometric pH probe in vivo, giving it potential for biological applications.

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noninvasive optical imaging because of their unique advantages, such as deep tissue penetration, minimum photodamage to biological samples, and less autofluorescence of biomolecules,17,18 which are favorable for real-time observing pH changes at intravital level to fit well in biomedical applications.19−21 In present NIR pH probes, most are based on photoinduced electron-transfer (PET) using NIR fluorophore, and they become highly fluorescent when the protonatable amino group within the fluorophore is protonated, but in this strategy, it is difficult to avoid the influence of probe concentration, probe environment and optical path length because of the single emission band.22 Fortunately, ratiometric fluorescent probes can be designed to effectively address these issues, which allow the measurement of changes of the intensity ratio of two emission bands induced by analyte and provide built-in correction for quantitative analysis. Therefore, several ratiometric NIR fluorescent pH probes have been proposed recently.23−26 However, cross talk between emission bands is the greatest challenge to design ideal ratiometric NIR probes, e.g., a newly ratiometric NIR probe for lysosomal pH

ntracellular pH is an important parameter associated with cellular behaviors and pathological processes, such as cell metabolism,1 proliferation, apoptosis,2 ion transport and homeostasis,3 enzyme activity,4 drug resistance5 and endocytosis.6 It is incontrovertible that the establishment and maintenance of an appropriate pH inside individual cellular compartments is of paramount importance to their normal physiology. However, disruption of normal cytoplasmic and organellar pH homeostasis or even slight changes would exert great influence to the progression of distinct pathophysiologic states and even lead to a diseased state, including cancer,7,8 cardiopulmonary and neurologic problems (such as Alzheimer’s disease).9,10 Usually, the cellular pH is carefully controlled at 7.2−7.4 in normal tissues,11 while the cellular pH gradient is substantially reduced or reversed in tumors compared to normal tissue. As a consequence, this difference can serve as a general tumor biomarker and provide an exploitable avenue for the treatment of cancer.12 Sensing extracellular/intracellular pH values and monitoring their fluctuation would provide indispensable information for further understanding the physiological and pathological processes closely relevant to pH. The requirements of intracellular pH sensing have driven the development of a number of molecular probes in the past.13−16 Among these, near-infrared (NIR) fluorescent probes that emit in the range of 650−900 nm are especially attractive for © XXXX American Chemical Society

Received: December 8, 2014 Accepted: January 20, 2015

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DOI: 10.1021/ac5045498 Anal. Chem. XXXX, XXX, XXX−XXX

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added into the above solution. The resulting solution was equilibrated for 5 min before measurement and then transferred to a quartz cell of 1.0 cm optical length. The fluorescence spectra were measured with excitation wavelength at 570 nm. The ratio signal (R = Ibasic/Iacidic) was calculated from the maximum fluorescence emission intensity Ibasic at basic conditions and Iacidic at acidic conditions. The slit width was 5 nm for excitation and emission. Cytotoxic Assays and Live Cell Imaging. The cytotoxic effect of NIR-Ratio was assessed using the MTT assay. Before imaging experiments, the cells were incubated with NIR-Ratio (10 μM, with 50% DMSO in the culture medium) for 45 min in the incubator at 37 °C with 5% CO2 and then rinsed for three times with phosphate buffered saline (PBS). For cell imaging at different pH, cells were further incubated at 37 °C for 15 min in high K+ buffer (30 mM NaCl, 120 mM KCl, 1 mM CaCl2, 0.5 mM MgSO4, 1 mM NaH2PO4, 5 mM glucose, 20 mM HEPES) with various pH values in the presence of 10 μM nigericin. To explore the effect of oxidative stress on pH fluctuations, HeLa cells were treated with redox active substance (H2O2/100 μM, NaClO/100 μM, NEM/1 mM, NAC/1 mM) for 1.0 h in PBS (20 mM, pH 7.4). Cells were treated to fluorescence imaging using an Olympus FV1000 laser confocal microscope. Fluorescence imaging was obtained by excitation with a multi Ar laser (565 nm). The fluorescence was collected in Channel 1 (650−700 nm) and Channel 2 (720− 760 nm), respectively, and analyzed by ImageJ software for pseudocolor ratio images. Animal Models and in Vivo Animal Studies. Nude mice (4 weeks old and about 15 g in weight) were purchased from SJA Co., Ltd. (Changsha, China). All animal operations were in accord with institutional animal use and care regulations, according to protocol No. SYXK (Xiang) 2008-0001, approved by Laboratory Animal Center of Hunan. Before animal imaging and analysis, nude mice were starved 12 h because of possible food fluorescence interference at the emission wavelength of the fluorescent dyes. We divided the mice into three groups A, B and C. To avoid occasionality, in each group we put three mice. For group A, lipopolysaccharide (2 mg/mL) in 100 μL PBS buffer solution was injected intraperitoneally (typically 0.2 mg, thus 0.012 mg/g of animal weight). For group B, NIRRatio-BTZ (250 μM) in 200 μL buffered solution was injected intraperitoneally. For group C, lipopolysaccharide (2 mg/mL) in 100 μL PBS buffer solution was injected intraperitoneally at first (typically 0.2 mg, thus 0.012 mg/g of animal weight). After 4 h, NIR-Ratio-BTZ (250 μM) in 200 μL of buffered solution was then injected in the same way. Before imaging, anesthesia of the mice was induced and maintained by inhalation of 5% isoflurane in 100% oxygen. The animals were placed into the imaging chamber, and kept under anesthetic using an isoflurane gas anesthesia system. Whole body images and NIR fluorescence images of nude mice were acquired using a Caliper VIS Lumina XR small animal optical in vivo imaging system. The imaging mode is set as excitation scan and Input/ Em was chosen as 570 nm for excitation, 650 and 720 nm for two different emission channels, respectively. All fluorescence images were acquired with auto exposure (FOV, 10 cm; f/stop 2; bin, high resolution), and fluorescence intensity was scaled as units of photons per second per centimeter square per steradian (ps−1 cm−2 sr−1).

measurement suffered serious spectral overlap (two emission peaks spacing is fixed at ∼34 nm),26 and therefore, it is difficult to provide high spatial resolution and sensitive information in fluorescence imaging. Further improvments in the imaging resolution will need the design of a ratiometric probe with less spectral overlap between two emission bands. To some extent, it is of great significance to develop a high resolution ratiometric NIR probe for determination of pH changes in live cells, tissues and organisms. Here, we report a series of ratiometric NIR fluorescent probes (NIR-Ratio series) based on hemicyanine skeleton with double near-infrared emission bands, whose emission properties and working pH ranges are distinct. Interestingly, the resolution of this probe between two emission bands is tunable by changing the substituent of the NIR conjugated structure, and the probes with different substituents show various red shifts from 31 to 76 nm between two NIR channels in response to pH changes. Among these, NIR-Ratio-BTZ modified with an electron-withdrawing substituent of benzothiazole showing two well-resolved emission peaks (the largest emission shift about 76 nm) can be considered as a good candidate for high resolution pH imaging in live cells and organisms. Moreover, NIR-Ratio-BTZ has an ideal pKa value (pKa ≈ 7.2) for monitoring the minor fluctuations of physiological pH near neutrality. With the help of laser scanning confocal microscope and small animal imager, the real-time ratiometric imaging and sensing of cellular pH in living HeLa cells and monitoring pH changes in living mice were achieved successfully by this ratiometric NIR probe, NIR-Ratio-BTZ.

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EXPERIMENTAL SECTION Chemicals and Instruments. All chemicals were obtained from commercial suppliers and used without further purification. Water used in all experiments was doubly distilled and purified by a Milli-Q system (Millipore, USA). Due to the somewhat solubility of NIR-Ratio series in water, stock solution (1 mM) of the probe was prepared by dissolving NIR-Ratio series in DMF. Different pH buffer solutions were prepared by using 50 mM potassium hydrogen phthalate (for pH 3.0−6.0 buffer), and potassium dihydrogen phosphate (for pH 6.1−9.0 buffer). The pH was adjusted by adding 0.1 M NaOH or 0.1 M HCl solutions. HeLa cells were obtained from the cell bank of Central Laboratory at Xiangya Hospital (Changsha, China). All experiments were carried out at room temperature. 1 H spectra were recorded on an Inova-400 spectrometer. Electrospray ionisation mass spectrometry (ESI-MS) analyses were performed by using a Waters Micromass ZQ-4000 spectrometer. UV−vis absorption spectra were measured on a Hitachi U-4100 spectrophotometer (Kyoto, Japan). Fluorescence intensity measurements were performed on a PTI QM4 Fluorescence System (Photo Technology International, Birmingham, NJ). pH value was measured by a model 868 pH meter (Orion). Fluorescent microscopy imaging of HeLa cells were carried out by using an Olympus FV1000-MPE multiphoton laser scanning confocal microscope (Japan). Fluorescent images of mice were taken by a Caliper VIS Lumina XR small animal optical in vivo imaging system (USA). In Vitro Fluorescent Detection. The stock solution of NIR-Ratio series (1 mM) was obtained by dissolving the material in DMF. One milliliter test solution of probe (1.0 μM) was made by introducing buffered dimethyl sulfoxide (DMSO) solution (20 mM standard buffer/DMSO = 9:1, v/v) to a centrifuge tube first and then 10 μL work solution of probe was B

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Analytical Chemistry Scheme 1. Structures of NIR-Ratio series and Its Response Mechanism to pH

Figure 1. Fluorescence emission spectra of NIR-Ratio series at acidic (dash line, pH 4.0) and basic (solid line, pH 8.0) conditions. (A) NIR-RatioBTZ. (B) NIR-Ratio-Cl. (C) NIR-Ratio-H. (D) NIR-Ratio-OMe.

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RESULTS AND DISCUSSION We designed and synthesized NIR-Ratio-H and its derivatives by introducing different substituents (such as methoxyl, chloride and benzothiazole) onto the hemicyanine skeleton. The derivatives were named NIR-Ratio-MeO, NIR-Ratio-Cl and NIR-Ratio-BTZ (Scheme 1), respectively. The details of synthetic routes of NIR-Ratio series outlined are shown in Scheme S1 (Supporting Information) and characterizations also are provided in the Supporting Information. We undertook the design with three aims in mind. First, the hemicyanine skeleton showed a significant NIR absorption and emission as reported,27 which is suitable for imaging in live tissues and living mice. Second, the ratiometric detection of pH would be feasible owing to that the intramolecular charge transfer (ICT) resulted from protonation/deprotonation at the hydroxyl in the

skeleton produces a shifted emission. Third, the emission wavelength and the pKa value can be easily modulated by introducing electron-donating or electron-withdrawing groups on the ortho-position of hydroxyl, which is conducive to screen the high resolution ratiometric probes. As a starting point, the change in the optical properties of NIR-Ratio-H with pH was first evaluated in buffer/DMSO solution (v/v = 9:1) after successful synthesization. As we had expected, NIR-Ratio-H exhibited emission maxima in the NIR region (650−760) both in acidic and basic media. As shown in Figures 1 and S1 (see the Supporting Information), under basic media (pH 8.0), NIR-Ratio-H has an absorption maximum (λabs) at 690 nm and a fluorescence emission maximum (λfl) at 708 nm. When the pH was changed from basic (8.0) to acidic (4.0), the λabs of NIR-Ratio-H was shifted to a shorter C

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Figure 2. DFT optimized structure of the protonation form (a) and deprotonation form (d) of NIR-Ratio-BTZ (in the ball-and-stick representation, carbon, nitrogen, oxygen and sulfur atoms are colored in gray, blue, red and yellow, respectively), and its molecular orbital plots HOMO (b, e); LUMO (c, f).

Figure 3. (A) Fluorescence emission spectra of NIR-Ratio-BTZ (1.0 μM) in buffer solution with pH changing from 3.0 to 9.0 (λex = 570 nm). (B) Linear relationship between I748/I672 (ratio of the fluorescence intensity of NIR-Ratio-BTZ at λ = 748 nm and λ = 672 nm) and pH values in the range pH 6.5−7.8. (C) Plots of Ibasic/Iacidic versus pH for NIR-Ratio-BTZ (I748/I672), NIR-Ratio-H (I708/I674), NIR-Ratio-OMe (I720/I689) and NIRRatio-Cl (I721/I677). (D) Time scan of fluorescence ratio I748/I672 of NIR-Ratio-BTZ in response to pH changes in buffer solution.

wavelength at 610 nm, accompanied by the color change from green to blue which could be easily distinguished by naked eye, and a new emission band at 674 nm appeared and underwent a concomitant increase with increasing hydrion concentration while the emission band at 708 nm weakened drastically (Figure S2, see the Supporting Information). These spectral shifts might be attributed to the protonation/deprotonation of the hydroxy group, thereby altering the intramolecular charge transfer (ICT). Owing to such sharp changes occurring at two different emission wavelengths simultaneously, NIR-Ratio-H possesses the basis for achieving NIR ratiometric detection of pH. However, the shift between two emission bands (Δλfl) is only 34 nm, which is not favorable for the high resolution imaging in living cells because of the serious cross talk. To address this issue, three analogues, NIR-Ratio-MeO bearing electron-donating substituent of methoxyl in the oposition of hydroxyl and NIR-Ratio-Cl, NIR-Ratio-BTZ bearing electron-withdrawing substituent of chloride and benzothiazole,

were synthesized and their spectroscopic properties were then investigated. As shown in Figure 1 and S1 (Supporting Information), all of them exhibited the similar spectra behavior toward acidic and basic conditions as NIR-Ratio-H; however, with the enhancement of ICT effect, the absorption and emission maxima showed an apparent bathochromic shift under basic condition but a hypochromatic shift under acidic condition, in other words, the Δλfl differed depending on the substituent. From Figure 1, we can see that the emission bands of NIR-Ratio-BTZ exhibited the largest bathochromic shift, ∼76 nm, with the pH change from acidic to basic, and NIRRatio-Cl had the second largest, but NIR-Ratio-MeO showed a negative change of the emission bands compared with NIRRatio-H. The shift of two emission bands (Δλfl) of NIR-RatioBTZ being much greater than other probes, can be attributed to the enhanced ICT between the donor and acceptor because of the strong electron-withdrawing group of benzothiazole, which D

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Analytical Chemistry Table 1. Photophysical Data for NIR-Ratio series compound

pH

Absmax(nm)

Emmax(nm)

ε (105 M−1 cm−1)

Φf

Δλfl (nm)

pKa

NIR-Ratio-BTZ

4.0 8.0 4.0 8.0 4.0 8.0 4.0 8.0

608 718 615 698 610 690 618 700

672 748 677 721 674 708 639 720

1.3 1.1 0.8 1.0 0.6 0.9 0.6 0.8

0.17 0.21 0.21 0.3 0.28 0.37 0.24 0.38

76

7.2

44

6.2

34

7.4

31

6.7

NIR-Ratio-Cl NER-Ratio-H NER-Ratio-OMe

when the pH value went from 3.0 to 9.0 and exhibited an excellent linearity in the pH range from 6.5 to 7.8 (Figure 3B), and the changing trend of absorption spectra were accordant (Figure S5, see the Supporting Information), meaning that the minor pH changes would not be omitted and ignored and important information about pH-affected physiology and pathology would be seized and stored. Therefore, of the four analogues used to detect pH, NIR-Ratio-BTZ has the best ratiometric responsive curve in the “neutral window”, which could be regarded as an ideal NIR probe for accurately monitoring the physiological pH near neutrality. Long-term stability of a pH probe is a crucial requirement for its practical applications in intracellular and in vivo imaging. The photostability of NIR-Ratio-BTZ was first measured by using a 150 W xenon lamp as an excitation source, and emissions at 672 and 748 nm in different pH media were recorded respectively during 2.5 h. It was clear that fluorescent signals collected at 672 and 748 nm were comparatively stable within the scanning period (Figure S6, see the Supporting Information). Moreover, a plot of fluorescence ratio (748 nm/ 672 nm) versus time was recorded during 1.5 h in which the pH of the buffer solution was adjusted every 15 min. From Figure 3D, the ratio changed fast and reached a plateau within a few seconds with the variation of pH value, which further indicated that NIR-Ratio-BTZ was only sensitive to pH change but stable to media, light and air. In addition, NIR-Ratio-BTZ displayed an excellent reversibility when the solution pH was circularly adjusted forth and back between 4.0 and 7.4 (Figure S7, see the Supporting Information). From these results, we could conclude that NIR-Ratio-BTZ possesses a fast and reversible response to H+ and exports a steady signal, which is favorable for real-time tracking the consecutive and frequent pH changes. To determine other interference on the pH measurement by biological molecules, the fluorescence spectra of NIR-RatioBTZ were measured in the presence of potential interferents under physiological conditions (pH 7.4, 37 °C), such as essential ions (Na+, K+, Ca2+, Mg2+, Zn2+, Fe3+, Fe2+, Ba2+, and Cu2+, as their chloride salts), and bioactive small molecules (glutathione (GSH), cysteine (Cys), homocysteine (Hcy), H2O2, and HClO). As shown in Figure S8 (see the Supporting Information), no significant variations in the fluorescent signals were observed in the presence of these biologically relevant species. On this basis, we propose that NIR-Ratio-BTZ has prominent selectivity to H+, enabling itself to be a promising pH-sensitive probe for studying pH-related biological processes without interference from the biological environment. The above results demonstrated that NIR-Ratio-BTZ has excellent selectivity and high sensitivity for detection of pH in buffer solution. However, complex biological samples usually contain endogenous components that will produce a high

might contribute to the high resolution ratiometric imaging toward pH for biological application. To provide a theoretical basis for understanding the effects of benzothiazole on the emission shift, density functional theory (DFT) calculations for NIR-Ratio-BTZ and NIR-Ratio-H with the B3LYP exchange functional employing 6-31+G(d) basis sets using a suite of Gaussian 09 programs were conducted. As shown in Figure 2, in the case of protonation, the dihedral angle between benzothiazole unit and hemicyanine skeleton in NIRRatio-BTZ is only 0.12°, which afforded a coplanar structure owing to the formation of strong O−H···N hydrogen bond (1.73 Å) between hydroxyl and nitrogen atom of benzothiazole. The hydrogen bond could induce the cationic character of the nitrogen in the benzothiazole, thereby increasing the electronwithdrawing ability. Consequently, the ICT within the hemicyanine is hampered. However, the deprotonation form of NIR-Ratio-BTZ displayed a large dihedral angle as 31.26°, demonstrating that benzothiazole unit and hemicyanine skeleton were not coplanar, which is favorable for the ICT effect. The obvious configuration changes have contributed to the large emission shift from acidic to basic condition. In addition, NIR-Ratio-BTZ possessed a HOMO−LUMO gap as 2.40 eV (protonation form) and 2.23 eV (deprotonation form) (Figure 2), whereas NIR-Ratio-H had the corresponding gap as 2.45 and 2.32 eV (Figure S3, see the Supporting Information), respectively. Therefore, the energy gap of NIR-Ratio-BTZ (0.17 eV) between protonation form and depronation form was larger than that of NIR-Ratio-H (0.13 eV), consistent with the observation that the larger emission shift is owned by NIRRatio-BTZ (76 nm) compared to NIR-Ratio-H (34 nm). Standard fluorescence pH titrations of NIR-Ratio series were then performed in buffer solution with different pH at a probe concentration of 1.0 μM. All of them displayed sensitive fluorescence spectroscopic response to the changes of pH values (Figures 3A, S2B, and S4, see the Supporting Information). With the pH increasing from 3.0 to 9.0, the emission band of them gradually bathochromically shifted with different extents, showing that the long-wavelength emission (Ibasic) remarkably raises while the short-wavelength emission (Iacidic) drops a lot. However, the quantitative analysis of the fluorescence ratio Ibasic/Iacidic vs pH showed different linear intervals because of the pKa values, which depends on the substituents (Figure 3C). The pKa values of NIR-Ratio series were derived from the titration curve of emission ratios (Ibasic/ Iacidic), and the pKa values were calculated to be 7.4, 6.7, 6.2 and 7.2 for NIR-Ratio-H, NIR-Ratio-MeO, NIR-Ratio-Cl and NIRRatio-BTZ (Table 1), which was almost identical to the linear response of the pH range, respectively. It was worth noting that the pKa value of NIR-Ratio-BTZ matched well with the normal physiological conditions (pH 6.8−7.4) of cytosol. Moreover, the ratio signal (I748/I672) of NIR-Ratio-BTZ increased 16-fold E

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Figure 4. Fluorescent images of HeLa cells clamped at pH 6.0, 6.5, 6.8, 7.2, 7.6 and 8.0, respectively. The images of the first and second row were collected in Channel 1 (650−700 nm) and Channel 2 (720−760 nm) with the excitation wavelength 565 nm, respectively. The third row exhibits the corresponding bright field images. The images of the fourth row (the ratio channel) were acquired by Image pro software. The color strip on the bottom shows the pseudocolor change with pH. Scale bar: 30 μm.

Figure 5. (A) Intracellular pH calibration curve of NIR-Ratio-BTZ (R indicates the pseudo ratio generated by Image pro software). The error bars show the standard deviation of the measurements. (B) Ratiometric images of HeLa cells loaded with NIR-Ratio-BTZ. Intact cells, and cells treated with 0.1 mM H2O2, 0.1 mM HClO, 1.0 mM NEM and 1.0 mM NAC, respectively. The ratiometric images were obtained from the green channel (650−700 nm) and red channel (720−760 nm) with the excitation wavelength 565 nm. (C) Intracellular pH values in the cells in panel B were estimated from their pseudo ratio and calibration curve. Scale bar: 30 μm.

autofluorescence background, and thus make the common fluorescent probes ineffective for assaying biotargets in these systems without sample pretreatment. Our designed probe NIR-Ratio-BTZ was based on NIR fluorescence, which makes it easy to discriminate the probe signal from the background fluorescence around 400−500 nm. To verify this, the RPMI1640 medium supplemented with 10% fetal bovine serum, was then used to investigate the feasibility of using this probe for detection of pH in biological samples. As shown in Figure S9 (see the Supporting Information), no obvious signal could be observed in the emission wavelength range from 650 to 820 nm in the absence of NIR-Ratio-BTZ. By contrast, upon addition of probe, a similar spectra but a slight red shift (about 15 nm) of

two emission peaks was observed compared with the spectra of NIR-Ratio-BTZ in buffer solution. The pH value was determined to be 7.38 ± 0.1 based on the calibration curve in Figure 3B, which was in excellent agreement with the measured value by pH meter. Most important, NIR-Ratio-BTZ was able to retain its significant fluorescence response to pH in cell culture medium. After having demonstrated in vitro that NIR-Ratio-BTZ is suitable for biological sample assay in a complex environment, we then explored its capacity for live cell imaging of pH changes. The cytotoxicities of NIR-Ratio-BTZ on HeLa cells were first evaluated using standard cell viability protocols (MTT assay). HeLa cells were treated with NIR-Ratio-BTZ for F

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whether it would be competent of fluorescence imaging in vivo and reflect occurrence of biological processes induced by exogenous stimulation. Therefore, we investigated the application of NIR-Ratio-BTZ to visualize pH changes in an abdominal inflammation model induced by lipopolysaccharide (LPS).31 The nude mice were divided into three groups, group one suffered an acute inflammatory response in peritoneal cavity via skin-pop injection of LPS as the control; group two was treated with saline simply in the peritoneal cavity, followed by intraperitoneal injection with NIR-Ratio-BTZ; group three first suffered an acute inflammatory response in the peritoneal cavity and then injected with NIR-Ratio-BTZ. The mice were imaged using a Caliper VIS Lumina XR small animal optical in vivo imaging system. As shown in Figure 6, the mouse treated

24 h with various concentrations (Figure S10, see the Supporting Information). We can see apparently that NIRRatio-BTZ (20 μM) would not cause distinct toxicity to cultured cell lines (survival rate is higher than 90%), indicating the low cytotoxicity and good biocompatibility of NIR-RatioBTZ. To prove NIR-Ratio-BTZ is suitable for pH quantification in live cells, the intracellular pH of HeLa cells was homogenized to the surrounding medium at varied pH values from 6.0 to 8.0 using the standard approach H+/K+ ionophore, nigericin,28 and the confocal fluorescence imaging was then carried out. As the pH value went up, the fluorescence emission in Channel 1 (protonation, 650−700 nm, Igreen) declined gradually while the fluorescence in Channel 2 (deprotonation 720−760 nm, Ired) grew higher (Figure 4), indicating less cross talk between the two emission channels. And the ratio imaging (Ired/Igreen, pseudocolor generated by ImageJ software) was obtained from the above two channels, which displayed a pH-dependent characteristic. Remarkable pseudocolor changes were observed between pH 6.0 and 8.0. Importantly, the fluorescence intensity ratio (Ired/Igreen) also exhibited a good linear calibration curve in the pH range from 6.5 to 8.0 as in vitro (Figure 5A), which was good for monitoring slight pH changes in physiological environments. Therefore, we next investigated the intracellular pH fluctuations influenced by oxidative stress. Different redox substances, such as H2O2, NaClO, NEM (N-ethylmaleimide, a GSH inhibitor) and NAC (N-acetylcysteine, a GSH precursor), were applied to treat HeLa cells and then these cells were imaged, respectively. As shown in Figure S11 (see the Supporting Information), compared with untreated cells, H2O2 caused a dramatic fluorescence increase in Channel 1 along with conspicuous fall in Channel 2, showing that intracellular pH was acidified by H2O2. This could be explained by that hydroxyl radicals produced by H2O2 generate some acidic substances such as phosphoric acid to cause acidification of cells.29 When HeLa cells were treated with NaClO, no obvious pH change was observed due to that the elevated level of ClO− could not increase greatly the intracellular acidic substances, which was consistent with the previous study under similar conditions.14b,16b In addition, it is reported that the decrease of GSH could affect the function of the Na+/H+ antiporter,30 herein, the GSH inhibitor NEM was used to incubate with HeLa cells to regulate the intracellular GSH level. From Figure S11 (see the Supporting Information), we can see that the fluorescence signal in Channel 1 and Channel 2 exhibited the same change tendency as treatment with H2O2, indicating that the depletion of GSH by NEM decreased the intracellular pH. However, the upregulation of GSH by NAC has no apparent effect on the intracellular pH, illustrating that GSH over the normal concentration may not cause a noticeable change of the intracellular acidic substances. The ratio imaging (Ired/Igreen) of HeLa cells treated with redox substances were obtained, as shown in Figure 5B. Based on the calibration curve derived from Figure 5A, the pH values for the untreated, H2O2, NaClO, NEM and NAC treated cells were determined to be 7.2 ± 0.05, 6.75 ± 0.1, 7.2 ± 0.06, 6.8 ± 0.08 and 7.3 ± 0.05, respectively (Figure 5C). These results demonstrated convincingly that our pH probe NIR-Ratio-BTZ displayed good performance with high contrast in intracellular imaging of pH and its fluctuations. Having achieved successful cell imaging with NIR-RatioBTZ, a further exploratory effort was made to determine

Figure 6. Representative fluorescence images (pseudocolor) of mice injected with NIR-Ratio-BTZ during LPS-mediated inflammatory response in vivo. (A) Only LPS was injected for control. (B) Saline was injected in the peritoneal cavity of mouse, followed by injection of NIR-Ratio-BTZ (50 μM). (C) LPS was injected into the peritoneal cavity of the mouse, followed by injection with NIR-Ratio-BTZ (50 μM). The mice were imaged with an excitation filter 580 nm and two emission channels of Channel 1 (650 nm) and Channel 2 (720 nm).

with saline and NIR-Ratio-BTZ exhibited low fluorescence intensity in Channel 1 (650 nm) and high intensity in Channel 2 (720 nm). However, the fluorescence intensity in the mouse treated with LPS and NIR-Ratio-BTZ changed evidently, a sharp increase in Channel 1 along with an obvious decline in Channel 2, which was attributed to that the inflammatory response could reduce the local pH to 5.5 or lower. As the control, the mouse treated with LPS but no NIR-Ratio-BTZ injection showed almost no fluorescence. Therefore, these results demonstrate that NIR-Ratio-BTZ is a prominent fluorescent probe for imaging pH changes in vivo.

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CONCLUSION In summary, we have developed a series of double NIRemissive ratiometric fluorescent probes (NIR-Ratio series) that exhibited pH-dependent optical responses. These hemicyanine derivatives, designed by a simple and flexible strategy, exhibited resolution-tunable characteristic showing various emission shifts from 31 to 76 nm between two NIR channels, and had G

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Analytical Chemistry different pKa values ranging from 6.2 to 7.4. The fluorescence behavior was explained by quantum chemical calculations with the B3LYP exchange functional employing 6-31+G(d) basis sets using a suite of Gaussian 09 programs. It was noteworthy that NIR-Ratio-BTZ, with a pKa value of 7.2, permits the real time monitoring of the near-neutral pH changes that is highly sensitive within the pH range of 6.5−7.8. Moreover, the large shift (Δλfl ≈ 76 nm) between two emission peaks, allowed NIR-Ratio-BTZ for quantitative measuring the pH value in cells with high resolution. Fluorescent imaging of pH and its fluctuations in vivo were also achieved successfully with the use of small animal imager, demonstrating its ability for detection of pH in complex biological systems. On the basis of these results, we therefore anticipate that our design strategy will be applicable to develop other high resolution probes for biological application, and envision that NIR-Ratio-BTZ may potentially be used as a clinically auxiliary tool for disease diagnosis and real-time monitoring of therapy effects.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details and additional spectroscopic data as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org

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AUTHOR INFORMATION

Corresponding Author

*R. Yang. E-mail: [email protected]. Fax: +86-73188822523. Author Contributions †

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge financial support through the National Natural Science Foundation of China (21305036, 21135001, 21405038 and J1103312), the Foundation for Innovative Research Groups of NSFC (21221003), the “973” National Key Basic Research Program (2011CB91100-0), Hunan Provincial Natural Science Foundation of China (2015JJ3035) and the Fundamental Research Funds for the Central Universities.

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