A Zero Cross-Talk Ratiometric Two-Photon Probe for Imaging of Acid

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A Zero Cross-Talk Ratiometric Two-Photon Probe for Imaging of Acid pH in Living Cells/Tissues and Early Detection of Tumor in Mouse Model Di Xu, Yinhui Li, Chung-Yan Poon, Hei-Nga Chan, Hung-Wing Li, and Man Shing Wong Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00520 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 2, 2018

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Analytical Chemistry

A Zero Cross-Talk Ratiometric Two-Photon Probe for Imaging of Acid pH in Living Cells/Tissues and Early Detection of Tumor in Mouse Model Di Xu,a, ‡ Yinhui Li,b, ‡, * Chung-Yan Poon,a Hei-Nga Chan,a Hung-Wing Li,a,* and Man Shing Wonga,* a

Department of Chemistry, Hong Kong Baptist University, Hong Kong, 999077, SAR China Key Laboratory for Green Organic Synthesis and Application of Hunan Province, Key Laboratory of Environmentally Friendly Chemistry, Application of Ministry of Education, College of Chemistry, Xiangtan University, Xiangtan, 411105, China. b

Email: [email protected]; [email protected]; [email protected]. Tel: 852-34117069, 852-34117065, 86-731-88822523

ABSTRACT: Acid-base disorders disrupt proper cellular functions which are associated with diverse diseases. Development of highly sensitive pH probes being capable of detecting and monitoring the minor changes of pH environment in living systems is of considerable interest to diagnose disease as well as investigate biochemical processes in vivo. We report herein two novel highresolution ratiometric two-photon (TP) fluorescent probes, namely PSIOH and PSIBOH derived from carbazole-oxazolidine π-conjugated system for effective sensing and monitoring acid pH in a biological system. Remarkably, PSIOH exhibited the largest emission shift of ~169 nm from 435 to 604 nm upon pH changing from basic to acidic with an ideal pKa value of 6.6 within a linear pH variation range of 6.2-7.0, which is highly desirable for high-resolution tracking and imaging the minor fluctuation of pH in live cells and tissues. PSIOH also exhibits high pH sensitivity, excellent photostability and reversibility as well as low cytotoxicity. More importantly, this probe was successfully applied to (i) sense and visualize the pH alteration in HeLa cells caused by various types of exogenous stimulation as well as; (ii) detect and differentiate cancer/tumor in liver tissues and mouse model, realizing its practical in vitro and in vivo applications.

Hydrogen ion is one of the most important intracellular species.1 Cell survival depends critically on the maintenance of a balanced pH,2 which modulates many metabolic pathways such as signaling, defense and apoptosis.3,4 Abnormal pH is thus often associated with malfunctions of organelles and many diseases including cancers.5-7 One of the characteristic features of tumor tissues is the acidic extracellular pH (pH 6.2-6.9) as compared to the normal tissues (pH 7.4),8 which is due to the increased glucose catabolism resulting in significant hydrogen ion production. The excess hydrogen ions diffuse from the tumor into adjacent normal tissue giving rise to a chronically acidic microenvironment of neighboring normal cells.9 Thus, there are tremendous interest to detect, monitor and track the distribution of abnormal pH fluctuation in living systems/tumor tissues to understand the pathological effects, the physiological functions as well as for early diagnosis of serious diseases. Fluorescence-based techniques are among the most powerful and non-invasive tools for their high sensitivity, fast response, convenience and unrivalled spatiotemporal resolution.10 On the other hand, the development of effective pH responsive fluorescent probes is indispensable for such detection and sensing.11 For now, most of pH responsive probes are based on either ratiometric or “turn-on” fluorescence measurement.12-16 The ratiometric approach, however, provides a more accurate quantitative analysis because of self-calibration and self-correction for external factors.17 The commonly adopted ratiometric detection mechanism is based on either intramolecular charge transfer system (ICT) or fluorescence resonance energy transfer (FRET).

There are however limitations for FRET on the spectral overlapping between donors and acceptors, while two ICT emission bands are still far from ideal that they usually suffer from serious cross-talk resulting in low spectral resolution in bioimaging and sophisticated data analysis. ICT-based probes with well-resolved emission peaks, which would allow precise and highresolution imaging for are still lacking. Ratiometric pH responsive imaging with two-photon excitation has become a very attractive tool to detect and monitor intracellular pH in living cells and tissues which can overcome limitations of one-photon excited ratiometric probes including high-energy/short-wavelength excitation that would cause photodamage and artificial production of reactive oxygen species (ROS) as well as allow only shallow tissue penetrating depth.18 Two-photon microscopy employing two near-infrared photons as the excitation source offers numerous merits including greater penetration depth (>500 µm), higher spatial resolution, and longer observation time. Nevertheless, practically useful high-resolution and sensitive TP ratiometric fluorescence probes for sensing and tracking intracellular acidic environment are rather rare.19-23 Despite a variety of pH sensing molecular probes reported so far, their applications on early tumor detection and growth monitoring in vivo has never been demonstrated. Herein, we report two novel high-resolution pH responsive TP fluorophores namely, PSIOH and PSIBOH (Figure 1). The applications of PSIOH in effective sensing and imaging the acidic environment in living cells as well as in detecting and monitor-

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Figure 1. (a) Molecular structures of PSIOH and PSIBOH. (b) pH sensing response of PSIOH.

ing cancer/tumor in tissues and mouse model has been demonstrated. In contrast to commonly employed pyridyl nitrogen atom or phenolic oxygen atom as a protonation/sensing site, these two ratiometric probes were developed from carbazoleoxazolidine π-conjugated system in which protonation triggers the oxazolidine ring-opening leading to an extension of π-conjugation and then greatly facilitating the intramolecular charge transfer (ICT) in the resulting fluorophore. Remarkably, the two interconvertible neutral and cationic (protonated) forms of these fluorophores exhibit an exceptionally large emission shift of 169-189 nm between these two states. Importantly, PSIOH affords a linear pH response range of 6.2 and 7.0, with an ideal pKa value of 6.60, suggesting that this probe is highly desirable for high-resolution sensing and imaging of the minor fluctuation of acidic cellular environment (intracellular pH = 6.8). Indeed, this probe was successfully applied to ratiometrically image and sense the changes of acidic environment in HeLa cells induced by various exogenous stimulations as well as detect and differentiate cancer/tumor in liver tissues by TP microscopy. Furthermore, realization of this probe for early tumor detection in vivo was first demonstrated in tumor mouse model.

EXPERIMENTAL SECTION Optical Measurements. Stock solution of PSIOH and PSIBOH (1.0 mM) was prepared in DMSO. One milliliter test solution of probe (10 μM) was made by introducing buffered dimethyl sulfoxide (DMSO) solution (DMSO : 0.2 M standard buffer = 1:9, v/v). In the pH titration experiments, working solution was poured into a quartz optical cell of 1 cm optical path length, and pH variation of the solution were achieved by adding the 0.2 M Na2HPO4-NaH2PO4 buffer and 0.1 M citric Acid-0.2 M Na2HPO4 buffer (for pH 3.40-9.2). The fluorescence spectra were measured with excitation wavelength at 320 nm and 380 nm, respectively. All spectroscopic experiments were carried out at room temperature. Determination of Quantum Yields. The quantum yields of PSIOH, SIOH and PSIBOH measured in DMSO and buffered solution (pH 3.0-9.2) were calculated by comparison with Norharmane (ΦR = 0.58 in 0.5 M H2SO4) and Rhodamine 6G (ΦR = 0.95 in Ethanol) as a reference using the following equation:24 Φ

Φ

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Where ΦF is the quantum yield, I is the integrated area under the fluorescence spectra, A is the absorbance, n is the refractive index of the solvent, and R refers to the reference fluorophore, Norharmane and Rhodamine 6G. Calculation of pKa Value. The pKa value of PSIOH and PSIBOH were estimated from the changes in the fluorescence intensity with various pH values by using the relationship, log[(Rmax - R)/(R - Rmin)] = pH - pKa. Rmin and Rmax are minimum and maximum limiting values of R, respectively. R is the ratio of emission intensity at two wavelengths. The pKa value was derived from plot of pH vs. log[(Rmax - R)/(R - Rmin)]. Cytotoxic Assays. The cytotoxic effect of PSIOH was assessed using the MTT assay. HeLa cells were first seeded into 96-well plates at a concentration of 4 × 103 cells/well in 100 μL of MEM medium with 10% FBS. Plates were maintained at 37 °C in a 5% CO2 95% air incubator for 24 h. After medium was removed, the HeLa cells were incubated with different concentrations (0, 10 nM, 1 μM, 10 μM and 50 μM) of PSIOH for 24 h. After that, PSIOH was replaced by fresh medium containing 0.5 mg mL-1 of MTT for 3 h with a dark incubation at 37 °C humidified incubator. The medium was replaced by DMSO to dissolve the formazan crystals and the plate was shaken gentle for 5 min. The absorbance was measured at 540 nm and 690 nm (as a reference) by Universal Microplate Reader. For data analysis, the mean values of the OD dye / OD control were calculated and error bars showed the standard deviation of three trials. Two-Photon Properties and Fluorescence Microscopy Imaging in Cells. Two-photon properties were measured by using femtosecond laser pulse generated by Ti: sapphire system (6001000 nm, 80 MHz, 140 fs) as an excitation source. Probe (1.0 × 10-6 M) was dissolved in PBS (20 mM, pH 7.0) containing 20% DMSO. The two-photon action cross-section (Φσ) spectrum of the probe were determined by the two-photon induced fluorescence method using a femtosecond pulsed laser in the range of 690−980 nm with Rhodamine 6G as the reference.25 The TPA cross-section was calculated by using the following equation: δ = δr(SsΦrфrcr) / (SrΦsфscs) Where s and r stand for the sample and reference molecules. S is the intensity of the signal collected by a CCD detector; Φ is the fluorescence quantum yield; c is the number density of the molecules in solution; and δr is the TPA cross section of the reference molecule. Fluorescence images of HeLa cells were obtained using an Olympus FV1000-MPE multiphoton laser scanning confocal microscope. The PSIOH-loaded cells were incubated at 37 °C for 15 min in high K+ buffer (120 mM KCl, 30 mM NaCl, 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, in which the use of nigericin was a standard protocol for homogenizing the pH of cells and culture medium to perform the intracellular pH calibration experiment. To explore the effect of oxidative stress on pH fluctuations, HeLa cells were treated with redox active substance (H2O2/0.1 mM, glucose/10 mM, and NH4Cl/1.0 mM), kept under oxygen content of 20%, 10%, 5% and 0% for 3 h and irradiated by UV light for 10 min and 30 min at 37 °C. Then, two-photon confocal fluorescence imaging of HeLa cells was observed under an Olympus FV1000-MPE multiphoton laser scanning confocal microscope, with a mode-locked titanium-sapphire laser source (120 fs, pulse width, 80 MHz repetition rate) set at wavelength of 750 nm.


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Two-Photon Fluorescence Microscopy Imaging in Liver Tissues. The slices were prepared from the liver of 14 day-old mice, and they were cut to 10 µm thickness by using a vibratingblade microtome in 25 mM PBS (pH 7.4). The slices were incubated with 10 μM PSIOH in PBS buffer bubbled with 95% O2 and 5% CO2 for 1 h at 37 °C, and then were washed three times with PBS, transferred to glass-bottomed dished, and observed under a two-photon confocal microscope (Olympus FV 1000-MPE). The 3D fluorescence images of the slices were acquired using 750 nm excitation and fluorescence emission windows of 400-450 nm and 590-630 nm. Animal Models and in Vivo Animal Studies. Athymic BALB/c nude mice were purchased from the Changsha SLAC Laboratory Animal Co. Ltd. and maintained under pathogenfree conditions. To examine whether PSIOH can in vivo detect the different pH in tumor region and normal region, 5 ×106 in vitro-propagated HeLa cells (in 100 μL of saline) were injected into the right rear leg region of 1820 g nude mice firstly. Once the tumor can be observed by naked eyes (about two weeks), the mouse was ready for in vivo imaging. After anesthesia, the right rear leg and the left rear leg of mouse (as control) were injected by 100 μL of 1 mg/mL PSIOH respectively. Then, the mouse was placed into the imaging chamber, and kept under anesthetic using an isoflurane gas anesthesia system. Whole body images were acquired using a Caliper VIS Lumina XR small animal optical in vivo imaging system. For further observing the pH change with the growth of tumor, the mouse was imaged again after four weeks. The imaging mode was set as excitation scan and Input/Em was chosen as 490 nm for excitation, 590-630 nm for emission channels, respectively.

RESULTS AND DISCUSSION Design, Synthesis and Characterization of Probe. Ratiometric fluorescent pH probes require a pH-responsive moiety in which protonation/deprotonation perturbs the π-conjugated system giving rise to a new and distinct emission. Among commonly employed pH-responsive moieties such as N-alkylated or amine group, the two emission peaks produced upon protonation and deprotonation are often not spectrally well-resolved which would lead to a severe cross-talk in emission and thus would not be useful for high-resolution imaging of extracellular microenvironment under minor pH variation. In addition, for sensitive and accurate analysis of the intracellular acidic conditions, it is essential for the probes to have a proper range of responsive pH. To search for a ratiometric probe with two highly resolved emissions and facile tunable pH working range to ideally suit for high-resolution imaging, we designed and developed carbazole-oxazolidine derived π-conjugated fluorophores namely, PSIOH and PSIBOH. These probes afford two highly resolved and reversible emission peaks upon protonation and deprotonation due to the greatly enhanced π-conjugation and ICT of the protonated fluorophore. In addition, the pKa value can be easily modified by introducing various electron-donating or electron-withdrawing group on the oxazolidine π-conjugated system. PSIOH and PSIBOH were synthesized by condensation of 1-(2-hydroxyethyl)-2,3,3-trimethyl-3H-indol-1-ium chloride and 3-(2-hydroxy ethyl)-1,1,2-trimethyl-1H-benzo[e]indol-3ium chloride with the corresponding carbazolecarbaldehyde and followed by the treatment with sodium hydroxide (Scheme S1), respectively. The chemical structures of the new probes were fully characterized by 1H NMR spectroscopy, 13C NMR spectroscopy, and high-resolution mass spectrometry (see SI) and agreed well with the proposed structures. It is important to

note that these carbazole-oxazolidine probes do not show photochromic effect under experimental conditions (Figure S1) even though some of 10-styrylindolino[2,1-b]oxazolidine derivatives were reported to undergo photochromism in acetonitrile solution under UV irradiation.26 Spectroscopic Properties and Optical Response towards Acidic pH. The optical properties of PSIOH, PSIBOH and their protonated forms, in buffer and DMSO are tabulated in Table S1. The optical responses of PSIOH and PSIBOH as ratiometric probes toward pH changes was investigated by the pH titration in DMSO-buffer solution (1:9, v/v) due to the low solubility of these neutral forms of carbazole-oxazolidine fluorophores in buffer only. When PSIOH was titrated from pH 9.2 to pH 4.5, the absorption at maximum, (λabs = 287 nm) gradually decreased concomitant with an increase in absorption at 340 nm and 491 nm (Figure S2). Meanwhile, the fluorescence titration showed a similar ratiometric behaviour with a progressive decrease of the emission, λem, at 435 nm and a simultaneous increase of the emission intensity at 604 nm upon excitation at 320 nm (Figure 2a). In addition, a distinct isoemission point at 543 nm was observed between two fully resolved λem at 435 and 604 nm (Figure 2a). As clearly seen in Figure 2b, the emission peak of this probe exhibited an exceptionally large emission shift with ∆λem ≈ 169 nm upon the pH change from basic to acidic. Such a dramatic spectral red shift is attributed to the ring-opening of the protonated oxazolidine moiety, giving rise to the enhancement of π-conjugation and ICT of the resulting protonated fluorophore, SIOH which would be potentially useful for high-resolution ratiometric imaging of pH variations in biological systems. Very similar absorption and emission spectral responses were observed for PSIBOH upon pH changes (Table S1, Figure S3a and Figure S3b). The pKa of the conjugate acid-base of PSIOH and PSIBOH was estimated to be 6.60 and 5.12 from the emission intensity ratios at λ = 604 and 435 nm (i.e. F604/F435), and λ = 619 and 430 nm (i.e. F619/F430), respectively,

Figure 2. Measurements were carried out in DMSO-buffer (0.2 M Na2HPO4, 0.2 M NaH2PO4) (1:9, v/v). (a) Emission spectra of PSIOH (10 μM) at different pH values in DMSO-buffer solution (1:9, v/v) (λex = 320 nm). (b) Fluorescence spectra of PSIOH at acidic (dash line, pH 4.49) and basic (solid line, pH 9.18) condition, respectively. (c) Emission intensity ratio (F604/F435) changes over the pH range of 4.5-9.2. The red line represents the nonlinear fitting of the experimental data by origin software. (Inset) Linear plot of F604/F435 against pH change between pH 6.2 and 7.0 (R = 0.9934). (d) Absorption and emission color changes of PSIOH in 10 μM of DMSO-buffer solution with pH 4.5 to 9.2 under an ambient and UV lamp light, respectively.



obtained in the fluorescence titration curve as shown in Figure 2c and Figure S3c. The smaller pKa value of PSIBOH was attributed to the basicity weakening caused by the incorporation of fused phenyl ring on the terminal indole moiety which shifts the electron density away from the nitrogen of oxazolo[3,2a]indole. It is important to note that the pKa value of PSIOH would be ideal for assessing and monitoring intracellular acidic environment in living cells and tissues. In addition, the F604/F435 ratio showed an excellent linearity against pH range between 6.2 and 7.0 with a linear coefficient of 0.9934 (Figure 2c, inset), indicating that PSIOH is versatile to quantitatively detect intracellular pH within such a range. Furthermore, the color change of PSIOH solution from pale yellow to orange upon a decrease in pH value (Figure 2d) can be readily visualized by naked-eyes. Importantly, such colorimetric transition was reversible with pH variations. Meanwhile, the fluorescence colour changing from blue to orange under UV lamp irradiation (365 nm) was also apparent upon shifting from basic to acidic. To understand the molecular and optical properties of PSIOH, PSIBOH and their protonated forms, the density functional theory (DFT) and time dependent density functional theory (TDDFT) calculations at the level of the B3LYP/6-31G* using Gaussian 09 software were performed.27 As shown in Figure S4, the π-electron on the highest occupied molecular orbital (HOMO) level of PSIOH primarily localized on the conjugated backbone of carbazole and the double bond whereas the electron density of the lowest unoccupied molecular orbital (LUMO) mainly localized on the double bond. In contrast, the π-electron on the HOMO level of SIOH was relatively stabilized and mainly localized on the electron-donating carbazole group whereas the π-electron on the LUMO level largely localized on the electron-deficient indolium moiety. Such electronic structures are highly favourable for the efficient intramolecular charge transfer from electron-donating carbazole moiety to electron-accepting indolium unit in the excited state. Consistently, similar electron density distribution were observed in PSIBOH. In addition, the HOMO-LUMO energy gaps of the cationic forms of PSIOH (1.89 eV) and PSIBOH (1.84 eV) are much narrower than those of neutral forms of PSIOH (2.35 eV) and PSIBOH (2.42 eV) as evaluated by TD-DFT calculations, indicating substantial red-shift of optical transition. Sensing Mechanism of Probe towards H+ Ion. This oxazolidine-based probe is anticipated to undergo acid catalyzed oxazolidine ring-opening in which C-O bond cleavage is facilitated by the protonation at the oxygen atom of the oxazolidine ring and then induced by the nucleophilic attack of nearby nitrogen atom. This would lead to the formation of cationic ICT state of the probe, which absorbs and emits at a longer wavelength than the neutral oxazolidine form, affording a H+ ion sensing response (Figure 1). To confirm the proposed sensing mechanism, 1 H NMR titration of PSIOH was conducted in a DMSO-d6 solution with a progressive addition of HCl. As shown in Figure 3, upon an increase in the amount of H+ ion, two diastereomeric methyl protons (h and g) on the oxazolo[3,2-a]indole ring of PSIOH gradually merge together and shift downfield. Meanwhile, the methylene protons, b, next to the N atom of the oxazolidine ring also show a dramatic downfield shift. The deshielding of this methylene protons (b) is due to the formation of iminium ion (C=N+) upon the opening of protonated oxazolidine ring. Furthermore, the formation of a strong ICT state in the protonated form causes the i and j protons on the double bond and k-q protons on the carbazole ring deshielded, leading to the downfield shift of these protons. In short, the proposed

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Figure 3. 1H NMR spectra of probe PSIOH (0.02 M) in DMSO-d6 upon the addition of H+: (a) free, (b) 0.25 equiv. of H+, (c) 0.5 equiv. of H+, (d) 0.75 equiv. of H+, (e) 1 equiv. of H+.

sensing mechanism was unambiguously demonstrated and ascertained in which PSIOH can instantly respond to the decrease in pH. Photostability, Reversibility, Selectivity, and Cytotoxicity of Probe. The excellent photostability of PSIOH under sensing conditions was confirmed as there were no significant difference in the relative fluorescence responses over a period of 2 h at pH 4.49, 6.64 and 8.04 (Figure S5a). PSIOH also exhibited a remarkable reversibility of pH switching between 4.49 and 9.18 which could rapidly complete within seconds (Figure S5b). This probe was found to be highly selective and specific towards pH change over other bioactive small molecules and metal ions. When various interference reagents at high concentration (50 μM) were added into the probe solution (10 μM) at pH 4.49, 6.64, and 8.04, respectively, no significant changes in fluorescence intensity ratio of the probe were observed (Figure S5c). Furthermore, PSIOH showed reasonably low cytotoxicity towards the HeLa cells with the lethal concentration 50 (LC50) of ~13 μM as determined by the MTT cytotoxicity assay (Figure S6). All these desirable properties are beneficial for the probe to be applied in biological applications. Ratiometric Two-Photon Fluorescence Imaging of Intracellular pH Change. Figure S7 shows the changes of the TP fluorescence spectra of PSIOH (350 M) at different pH values in DMSO-buffer (1:9, v/v) with λex = 750 nm. As seen, the spectral emission wavelengths and behaviour upon titration are relatively comparable to those obtained by one-photon excitation counterpart (Figure 2a) except the fluctuated emission intensity below 400 nm due to the poor detector sensitivity below 400 nm of femtosecond laser system that we used. Nevertheless, it does not affect two photon imaging studies and results in which the blue channel was collected at 400-450 nm. The TP action cross-section spectrum of PSIOH as determined by the two-photon induced fluorescence method was first evaluated for its potential use in TP microscopic imaging. Despite such a short π-conjugated backbone, the two-photon action cross-section measured was ~55 GM at 750 nm (Figure S8), which would be sufficient to provide good contrast and brightness for ratiometric two-photon bio-images. To demonstrate the application of PSIOH to detect and sense the pH change of intracellular environment, TP microscopic imaging study was carried out using HeLa cell with H+/K+ ionophore, nigericin, serving to homogenize the pH of cells and culture medium.28 Upon excitation at


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750 nm, the fluorescence inside the cells increases upon an increase in pH value in the blue channel (second row) whereas the fluorescence in the red channel (third row) decreases in intensity (Figure S9). It is worth mentioning that PSIOH shows a fast response to the change in intracellular pH and distributes relatively evenly within the cell without a subcellular localization selectivity as shown in the overlay of bright-field and TP fluorescence images (fourth row), which is different from most of molecular TPM probes reported. Because of better biocompatibility and dispersity, PSIOH thus could be used to measure and map the intracellular pH within the entire living cell rather than a particular organelle. Tracking the Stimulated Intracellular pH Fluctuation by PSIOH via TPM Fluorescence Imaging. To investigate the capability of this probe in monitoring intracellular acidicity/pH alteration, different exogenous stimulants were adopted to induce intracellular pH fluctuation. We here employed (i) redox chemicals such as H2O2 and glucose to modify the ion transport activity of cell membrane resulting in acidification of intracellular pH to 7.0 ± 0.2;29 and (ii) ammonia chloride to induce the pH changes of lysosomes inside the cell from 4.7-4.8 to 6.26.4.30 As shown in Figure 4a, H2O2, glucose and NH4Cl induce pH change in the intracellular environment of HeLa cells to different extent. The overlay images clearly show the difference in fluorescence intensities in the red and blue channels ascertaining the ability of PSIOH in responding to the exogenous stimulants that induced intracellular pH change accordingly. In hypoxia (i.e. oxygen deficiency), the mitochondrial oxygen consumption rate and production of ATP are reduced, which decreases cytosolic pH resulting in intracellular acidosis.31 Thus, PSIOH was also used to track the intracellular pH change upon a change of oxygen content (20%, 10%, 5% and 0% of O2) in HeLa cells. With a reduction of oxygen content (Figure 4b), much higher fluorescence intensities were observed in the red channels as compared to those from the blue channels. Importantly, this probe showed distinct fluorescence intensity differences from the two channels under different oxygen level in the cells. This result implies that PSIOH is highly sensitive to an intracellular pH change induced by insufficient oxygen conditions. On the other hand, it is known that reactive oxygen species (ROS) such as hydrogen peroxide may damage H+-ATPase in plasma membrane causing the intracellular environment more acidic. As a demonstration, PSIOH was applied to track the intracellular pH change as induced by ROS. The PSIOH-loaded HeLa cells were subject to irradiation with UV light over a period of time to cause the generation of hydrogen peroxide leading to an increase in intracellular acidity. As shown in Figure S10, the un-irradiated cells exhibited a strong fluorescence in the blue channel. On the other hand, the fluorescence signal in the red channel becomes more and more intense as the UVexposure time gets longer. These results clearly indicate that PSIOH can rapidly and distinctly respond to the change to the intracellular acidic environment induced by ROS production. Two-Photon Ratiometric Fluorescence Imaging of pH in Living Liver Slices. To demonstrate the advantage of using PSIOH for deep tissue imaging of abnormal pH variation, twophoton ratiometric imaging of PSIOH in live cancer and normal mice liver tissue slices was carried out. Figure 5 clearly shows a distinct difference in the ratiometric fluorescence pattern between cancer and normal mice liver tissues in which the cancer

Figure 4. (a) TP microscopic images of PSIOH loaded HeLa cells. Intact cells, H2O2 (0.1 mM) treated, glucose (10 mM) treated and NH4Cl (1.0 mM) treated cells were incubated 1 h at 37 °C. (b) TP microscopic images of PSIOH loaded HeLa cells were kept under 20%, 10%, 5%, and 0% oxygen content for 3 h at 37 °C. All the images were obtained from blue channel at 400-450 nm and red channels at 580-620 nm with λex = 750 nm. The fourth rows of (a) and (b) are pseudo-colored ratiometric images constructed from blue and red channels, respectively.

liver tissue exhibits much stronger fluorescence in the red channel than in the blue channel with an average pH value of ~6.54. On the other hand, the normal liver tissue shows strong blue fluorescence but weak red fluorescence with an estimated pH value of ~7.39. These findings suggest that PSIOH is a versatile and useful tool to differentiate cancer tissue from normal one. Furthermore, the confocal Z-scan two-photon imaging sections at different penetration depth (0-172 μm) were recorded and depicted in Figure S11. Consistently, the fluorescence signal of cancer liver tissue was much stronger in red channel than in blue channel down to a depth of 172 μm as compared with those of normal liver tissue, which further suggested that this TP probe



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mal section of mice (green circle). Furthermore, the fluorescence intensity of tumor section after 4 weeks of induction was much stronger than that of the 2-week post-induction. These results unambiguously demonstrated that the PSIOH probe was highly sensitive to detect the weakly acidic environment of tumor tissue in the early stage. As the growth of the tumor continued, the more extensive the acidic environment would be, the stronger fluorescence signal was thus observed in the tumor section after 4-week injection. As a result, this probe would be practically effective and promising as an early diagnostic tool for tumor detection as well as monitoring and tracking various tumor stages in vivo.

CONCLUSIONS Figure 5. TP ratiometric fluorescence images of pH in cancer and normal mouse liver slices by loading with 20 μM probe for 2 h. The TP images were obtained from blue (400-450 nm) and red (580-620 nm) channels with λex = 750 nm. The third column is pseudo-coloured ratiometric images constructed from blue and red channels. Scale bar: 50 μm.

can effectively detect the pH variation in deep tissue imaging of live mice tissue using TPM. In Vivo Fluorescence Imaging of PSIOH in Tumor Mouse Model. In view of the high pH sensitivity and ideal pKa value of PSIOH, its practical application in early detection of tumor in mouse model was then investigated. PSIOH was injected via intratumoral injection to the left leg of mice in which they were injected with HeLa cells 2 and 4 weeks ago, respectively to induce tumor growth. As shown in Figure 6, the fluorescence signal collected at 580-620 nm was clearly brighter in tumor section (red circle) after 2 weeks of induction than that of the nor-

In summary, we have developed two carbazole-oxazolidine derived fluorophores, namely PSIOH and PSIBOH as novel and effective ratiometric two-photon probes for pH sensing and imaging based on the protonation induced oxazolidine ring-opening mechanism. Because of the exceptionally large shift in the emission bands (169-189 nm) of the two emissive states upon protonation/deprotonation, these TP ratiometric probes can be applied for a high-resolution sensing and imaging of pH changes in cellular microenvironment. Remarkably, PSIOH has been found to show an ideal pKa value of 6.60 for detection and monitoring acid pH changes in intracellular/extracellular environment of living cells and tissues via TP microscopy. This twophoton ratiometric sensing probe was also found to exhibit high signal-to-noise ratio, excellent photostability and reversibility as well as low cytotoxicity. Exceptionally, this probe not only senses and visualizes various types of exogenous stimulation that caused acid pH change in HeLa cells but also shows medically relevant applications in detecting and differentiating cancerous cells in liver tissues and tumor in mouse model. This is the first and effective pH responsive molecular probe for early detection and diagnosis of tumor in vivo.

ASSOCIATED CONTENT Supporting Information Experimental details and additional spectroscopic data as noted in text. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author * Email: [email protected]. Fax +86-731-88822523 * Email: [email protected]. Fax +852-34117065 * Email: [email protected]. Fax +852-34117069

Author Contributions ‡These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT Figure 6. Tumor-bearing mice were injected with PSIOH via intratumoral injection after 2 and 4 weeks of tumor growth and then imaged, respectively. The green circle indicated the position of the normal tissue. The red circle indicated the position of tumor injected. The mice were imaged with an excitation filter 490 nm and the emission was collected at 580-620 nm. The colored scale indicates the relative fluorescence intensity.

This work was supported by Hong Kong Research Grant Council (GRF 12301317), the National Natural Science Foundation of China (No. 21675135), 2014 Hong Kong Scholars Program, and Institute of Molecular Functional Materials, which was supported by a grant from the University Grants Committee, Areas of Excellence Scheme (AoE/P-03/08).

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