Highly Specific and Ultrasensitive Two-Photon Fluorescence Imaging

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Highly Specific and Ultrasensitive Two-photon Fluorescence Imaging of Native HOCl in Lysosomes and Tissues Based on Thiocarbamate Derivatives Baocun Zhu, Ping Li, Wei Shu, Xin Wang, Caiyun Liu, Yue Wang, Zuokai Wang, Yawei Wang, and Bo Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04392 • Publication Date (Web): 25 Nov 2016 Downloaded from http://pubs.acs.org on November 27, 2016

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Analytical Chemistry

Highly Specific and Ultrasensitive Two-photon Fluorescence Imaging of Native HOCl in Lysosomes and Tissues Based on Thiocarbamate Derivatives Baocun Zhu,†,‡,§ Ping Li,†,§ Wei Shu,‡ Xin Wang,† Caiyun Liu,‡ Yue Wang,‡ Zuokai Wang,‡ Yawei Wang,‡ Bo Tang*,† †

College of Chemistry, Chemical Eng ineering and Materials Science, Co llaborative Innovation Center of Functionalized Probes for Chemical Imaging in Un iversities of Shandong, Key Laboratory of Mo lecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Institute of Bio medical Sciences , Shandong Normal Un iversity, Jinan 250014, P. R. China. Fax: (86)531 86180017. E-mail: [email protected]. ‡ School of Resources and Environment, University of Jinan, Shandong Provincial Engineering Technology Research Center for Ecological Carbon Sink and Capture Ut ilizat ion, Jinan 250022, China.

ABSTRACT: Hypochlorous acid (HOCl) acts as a weak acid distributed mainly in acidic organelle lysosomes of phagocytes and plays crucial roles in the immune defence. The elaborate interrelation between the variations of HOCl levels in lysosomes and different physiological and pathological processes remains unclear. Thus, the accurate determination of lysosomal HOCl in living cells and in vivo is very important. Because of extremely low concentration and difficult to distinguish from OCl- under the physiological environment, it is still a great challenge to specifically monitor the intracellular intrinsic HOCl levels without exogenous stimulation, which impedes an exact understanding of its biological roles. In this paper, based on the electrophilic addition of Cl+ to sulfide moiety, we have developed a two-photon fluorescent probe O-(N-butyl-1,8-naphthalimide)-4-yl-N,N-dimethylthiocarbamate (NDMTC) for the specific determination of HOCl over OCl- and other bioactive molecules. Our results show that NDMTC possesses a detection limit of 7.6 pM, and is the first fluorescent probe for detecting HOCl at the picomolar level. Furthermore, by introducing an alkylmorpholine group to NDMTC framework, the lysosome-targetable derivative Lyso-NDMTC was obtained, and its ability to image HOCl in the lysosome organelles was clearly confirmed. Combined with two-photon fluorescence imaging of background suppression and deeper tissue penetration, NDMTC and Lyso-NDMTC were used to successfully visualize intracellular native HOCl and discern tumor tissues in mice. This study offers two perfect fluorescence imaging probes for further investigation of pathological roles of HOCl in various diseases.

the variations of HOCl levels in lysosomes and different physiological and physiological processes remains unclear because of a lack of sensitive imaging technique for monitoring lysosomal native HOCl in living cells and in vivo. Fluorescence imaging has been widely applied to living systems for disclosing cellular functions of biologically active molecules owing to its high temporal and spatial resolution.1116 Although various fluorescent probes for HOCl have been developed, available fluorescent probes can exclusively detect the increased concentration HOCl by external stimulation. This is because their unsatisfied sensitivity and very low actual concentration of HOCl in living system.17-31 Additionally, the strict distinction between HOCl and OCl- has not been taken into account in the previous studies, even though HOCl coexisted with OCl- under the physiological condition.17-31 Thus, it is still a great challenge to specifically

INTRODUCTION Hypochlorous acid (HOCl), one of the important reactive oxygen species (ROS), can be produced from H2O2 and Clwith the assistance of myeloperoxidase (MPO) in living systems.1 It acts as a weak acid (pKa of 7.6 for HOCl) distributed mainly in acidic organelles lysosomes of macrophages,2,3 and plays crucial roles in the immune defence against invasive bacteria and other pathogens despite extremely low concentration.4,5 On the other hand, excessively produced HOCl in phagocytes is associated with a wide variety of diseases.6,7 For example, aberrant accumulation of HOCl in lysosomes could induce chronic disease resulted from the apoptosis of cultured cells through the rupture of lysosomes.8 Additionally, recent evidences demonstrate lysosomal functions are subtly regulated by ROS including HOCl.9,10 Unfortunately, up to now, the elaborate interrelation between

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monitor the intracellular native HOCl levels without exogenous stimulation. Two-photon microscopy (TPM) possesses unique advantages such as deeper tissue penetration, low phototoxicity, and three-dimensional (3D) imaging of living tissues.32-36 More importantly, TPM collocated with the longwavelength excitation is a very important way of enhancing the detection sensitivity in the bioimaging applications benefiting from reduced background fluorescence. Thus, in consideration of necessity and urgency to track the intrinsic HOCl levels of lysosomes in living cells and in vivo, novel lysosome-targetable two-photon fluorescent probes for the ultrasensitive and specific imaging of intracellular native HOCl became our targets. Herein, we present a new strategy for the design of HOClspecific fluorescent probes employing thiocarbamate as a recognition receptor based on the electrophilic addition of Cl+ to sulfide moiety response mechnism. The as-obtained twophoton fluorescent probe O-(N-butyl-1,8-naphthalimide)-4-ylN,N-dimethylthiocarbamate (NDMTC) exhibits excellent sensitivity and high specificity for HOCl over OCl- and other biological active molecules at higher concentrations. NDMTC could be successfully applied to tracking the fluctuation of endogenous HOCl levels in live macrophage cell line (RAW 264.7) and discriminating cancer cells from normal cells. Subsequently, based on the molecular framework of NDMTC, its lysosome-targetable derivative Lyso-NDMTC was developed by introducing an alkylmorpholine group and it applications to image intracellular HOCl in lysosomes was nicely demonstrated. Finally, these two fluorescent probes NDMTC and Lyso-NDMTC were applied to discriminating tumor tissues from normal tissues in mice. Scheme 1. The synthesis of fluorescent probes NDMTC, Lyso-NDMTC, NDMC, and NDETC

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column chromatography (dichloromethane as eluent) to get the pure probe NDMTC (132.6 mg, 37.2%). 1H-NMR (400 MHz, DMSO-d 6) δ (*10-6): 0.934(t , J = 7.4 Hz, 3H), 1.3171.410(m, 2H), 1.585-1.659(m, 2H), 3.444(s , 3H), 3.519(s , 3H), 4.045(t , J = 7.4 Hz, 2H), 7.594(d , J = 8.0 Hz, 1H), 7.880(t , J = 7.8 Hz, 1H), 8.286(d, J = 8.4 Hz, 1H), 8.502-8.526(m, 2H). 13C-NMR (100 MHz, DMSO-d 6) δ (*10-6): 14.19, 20.28, 30.10, 43.59, 120.26, 121.92, 122.86, 126.13, 128.02, 128.92, 131.57, 131.61, 154.87, 163.23, 163.74, 185.85. ESI-MS calcd for C19H21N2O3S [M+H]+ 357.1273, found 357.1259. Synthesis of probe Lyso-NDMTC The lysosome-targetable fluorescent probe Lyso-NDMTC was prepared refering to the preparation procedure of NDMTC. Typically, N-morpholinoethylamino-4-hydroxy-1,8naphthalimide (326.3 mg, 1.0 mmol) and dimethylthiocarbamoyl chloride (618.0 mg, 4.0 mmol ) were put in dry dichloromethane (10.0 mL). Next, Ndiisopropylethylamine (350.0 μL) was added to the abovementioned reaction solution. The reaction was carried out at room temperature until it completed. Finally, the pure probe Lyso-NDMTC was obtained (137.3 mg, 33.2%). 1H-NMR (400 MHz, DMSO-d 6) δ (*10-6): 3.538(b r, 6H), 3.645(d , J = 12 Hz, 2H), 3.765(t , J = 12.0 Hz, 2H), 3.445(s , 3H), 3.524(s , 3H), 4.442(t , J = 6.0 Hz, 2H), 7.618(d , J = 8.0 Hz, 1H), 7.904(t , J = 8.0 Hz, 1H), 8.315(d, J = 8.4 Hz, 1H), 8.532 (d , J = 8.0 Hz, 2H). 13C-NMR (100 MHz, DMSO-d 6) δ (*10-6): 18.40, 34.60, 41.97, 43.61, 51.77, 53.68, 54.10, 63.55, 120.45, 121.96, 123.06, 126.16, 128.04, 129.15, 129.21. 131.65, 131.72, 155.01, 163.79, 164.34, 185.80. ESIMS calcd for C21H24N3O4S [M+H]+ 414.1488, found 414.1485. Synthesis of control compound NDMC The control compound O-(N-butyl-1,8-naphthalimide)-4-ylN,N-dimethylcarbamate (NDMC) was prepared refering to the preparation procedure of NDMTC. In brief, N-butyl-4hydroxy-1,8-naphthalimide (269.3 mg, 1.0 mmol) and dimethylcarbamic chloride (322.5 mg, 3.0 mmol ) were put in dry dichloromethane (10.0 mL). And then, Ndiisopropylethylamine (400.0 μL) was added to the abovementioned mixture. Finally, the control compound NDMC was obtained (210.4 mg, 61.8%). 1H-NMR (400 MHz, CDCl3) δ (*10-6): 0.980(t , J = 7.4 Hz, 3H), 1.404-1.497(m, 2H), 1.692-1.754(m, 2H), 3.113(s , 3H), 3.303(s , 3H), 4.181(t , J = 7.6 Hz, 2H), 7.595(d, J = 8.0 Hz, 1H), 7.765(t , J = 8.0 Hz, 1H), 8.310(d, J = 8.4 Hz, 1H), 8.594-8.630(m, 2H). 13C-NMR (100 MHz, CDCl3) δ (*10-6): 13.86, 20.38, 30.20, 36.77, 37.05, 40.24, 119.21, 119.68, 122.88, 125.56, 126.95, 127.82, 129.28, 131.48, 131.91, 152.37, 153.60, 163.60, 164.16. ESI-MS calcd for C19 H21N2O4 [M+H]+ 341.1501, found 341.1534. Synthesis of probe NDETC The compound O-(N-butyl-1,8-naphthalimide)-4-yl-N,Ndiethylthiocarbamate (NDETC) was prepared refering to the preparation procedure of NDMTC. Generally, N-butyl-4hydroxy-1,8-naphthalimide (269.3 mg, 1.0 mmol) and diethylcarbamic chloride (455.1 mg, 3.0 mmol ) were put in dry dichloromethane (10.0 mL). Next, Ndiisopropylethylamine (200.0 μL) was added to the abovementioned reaction solution. Finally, the pure probe NDETC

EXPERIMENTAL SECTION Synthesis of probe NDMTC N-butyl-4-hydroxy-1,8-naphthalimide (269.3 mg, 1.0 mmol) and dimethylthiocarbamoyl chloride (618.0 mg, 5.0 mmol) were put in dry dichloromethane (10.0 mL). Subsequently, Ndiisopropylethylamine (400.0 μL) was added to the abovementioned solution. The resulting mixtures were stirred at room temperature until the reaction completed. After removal of dichloromethane, the residues were purified through silica

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was obtained (229.8 mg, 59.8%). 1H-NMR (400 MHz, CDCl3) δ (*10-6): 0.982(t , J = 7.2 Hz, 3H), 1.375-1.442(m, 4H), 1.442-1.492(m, 4H), 1.674-1.750(m, 2H), 3.8483.902(m, 2H), 3.931-3.985(m, 2H), 4.185(t, J = 7.6 Hz, 2H), 7.456(d , J = 8.0 Hz, 1H), 7.762(t , J = 8.0 Hz, 1H), 8.168(d , J = 8.0 Hz, 1H), 8.605-8.631(m, 2H). 13C-NMR (100 MHz, CDCl3) δ (*10-6): 11.77, 13.84, 13.87, 20.39, 30.21, 40.25, 44.75, 48.71, 120.48, 121.03, 123.11, 126.28, 127.15, 128.19, 129.30, 131.50, 131.55, 154.81, 163.54, 164.09, 185.83. ESI-MS calcd for C21H25N2O3S [M+H]+ 385.1586, found 385.1583.

structure (Fig. S3). To the best of our knowledge, NDMTC was the first fluorescent probe for the quantitative analysis of HOCl at the picomolar level.

RESULTS AND DISCUSSION Design, synthesis and properties of fluorescent probe NDMTC for HOCl. To design highly selective fluorescent probes for HOCl, specific reaction between recognition group and HOCl is a crucial. Previous reports suggest that HOCl can oxidize sulfide-type amino acids through the initiation of an electrophilic addition of chlorinium ion (Cl+ ) from the decomposition of HOCl.37,38 This feature of HOCl is different from other ROS including OCl- and reactive nitrogen species (RNS). Therefore, we envisioned that N,N-dimethylthiocarbamate (DMTC) containing sulfide moiety might be an ideal receptor for HOCl over other biological active molecules, especially OCl-. Furthermore, in view of the intramolecular charge transfer (ICT) strategy, the generation of “push-pull” fluorophore structure induced by the specific reaction of HOCl was adopted to enhance the sensitivity.39-41 In 2011, 4-hydroxy-1,8naphthalimide with a highly effective “push-pull” (oxygen anion-imide) structure was employed to construct an ICTbased fluorescent probe for the first time by us.42 It has been widely used in the construction of fluorescent probes owing to its excellent photophysical properties including large twophoton absorption cross section.43-45 Hence, we prepared fluorescent probe O-(N-butyl-1,8-naphthalimide)-4-yl-N,Ndimethylthiocarbamate (NDMTC) through a simple esterification between 4-hydroxy-1,8-naphthalimide and dimethylthiocarbamoyl chloride (Scheme 1). The spectroscopic properties of NDMTC were studied in the simulated physiological conditions (PBS, 5.0 mM, pH = 7.4). NDMTC exhibited very weak fluorescence in the absence of HOCl at the excitation of 410 nm (one-photon excitation wavelength) and 800 nm (two-photon excitation wavelength), which was attributed to the highly effective inhibition of ICT structure.46,47 Interestingly, the introduction of HOCl into the solution of NDMTC resulted in an intensive fluorescence enhancement at 547 nm (Fig. S1). Additionally, a good linear relationship between the fluorescence intensities at 547 nm and the HOCl concentrations (1.0 ~ 20.0 μM) was obtained (Fig. S2). To evaluate the feasibility of NDMTC for detecting extremely low concentration HOCl, the titration of HOCl at lower concentrations was performed. As expected, there is a satisfactory linear relationship between the fluorescence intensities at 547 nm and the HOCl concentrations (0 ~ 1500.0 pM), and the detection limit of 7.6 pM was calculated by equation DL = 3σ/k (Fig. 1). The ultrasensitivity of NDMTC was ascribable to high reactivity of DMTC recognition group and the adoption of sensitive 4hydroxy-1,8-naphthalimide fluorophore with strong ICT

Figure 1. The fluorescence spectra of NDMTC (5.0 μM ) with the addition of different concentrations of HOCl (0 ~ 1500.0 pM ) under PBS/ethanol (1:1, v/v, pH = 7.4) buffer solution. λex = 410 nm, slit widths: Wex = Wem = 5 nm. The inset is the fluorescence intensities at 547 nm vs increasing concentrations of HOCl. Each datum was acquired 10 min after HOCl addition.

Then we examined the specificity of NDMTC towards HOCl. As shown in Fig. 2, NDMTC did not display obvious fluorescence spectra changes in the presence of other ROS, RNS, biothiols, metal ions, and amino acids, including H2O2, O2-, tert-butylhydroperoxide (TBHP), tert-butoxy radical (·OtBu), hydroxyl radical (·OH), 1O2, NO2-, NO3-, NO, peroxynitrite (ONOO-), glutathione (GSH), K+ , Ca2+ , Na+ , Mg 2+ , Zn 2+ , glutamic acid (Glu), alanine (Ala), valine (Val), threonine (Thr), lysine (Lys), leucine (Leu), and ascorbic acid (AA) at higher concentrations (500.0 μM). To confirm the recognition capability of NDMTC for HOCl rather than OCl-, absolute ethanol was chosen as the medium to avoid the mutual-transformation of HOCl and OCl- with the aid of superfluous water. As expected, the introduction of HOCl into the solution of NDMTC could result in the dramatic fluorescence enhancement, but OCl- did not (Fig. S4). To furhter verify the selectivity of NDMTC for HOCl over OCl-, the experiments were carried out in the completed water solution. The analytical conditions at pH = 2.0 and pH = 12.0 were chosen for hypochloric species nearly appeared in a state of HOCl at pH = 2.0 and OCl- at pH = 12.0. The results exhibited that NDMTC could detect selectively HOCl rather than OCl- (Fig. S5). In addition, the capabilities of NDMTC for detecting HOCl at different pH buffer solution were investigated in detail (Fig. S6). It can be seen that NDMTC was stable in the range of pH 2.0 – 10.0, and its ability of recognition for HOCl was nearly invariant. Collectively, these results clarified that NDMTC is a specific fluorescent probe for HOCl, and may accurately detect intracellular HOCl without interference from other bioactive molecules including OCl-.

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Figure 2. The fluorescence responses of NDMTC (5.0 μM ) towards HOCl (10.0 μM ) and other various analytes (500.0 μM except 3000.0 μM GSH) under PBS/ethanol (1:1, v/v, pH = 7.4) buffer solution. a: Blank, b: H 2O2, c: O 2-, d: TBHP, e: ·O tBu, f: ·OH, g: 1O2, h: NO 2-, i: NO 3-, j: NO, k: ONOO -, l: GSH, m: K + , n: Ca2+ , o: Na+ , p: M g2+ , q: Zn2+ , r: Glu, s: Ala, t: Val, u: Thr, v: Lys, w: Leu, x: AA, and y: HOCl. λex = 410 nm, slit widths: Wex = Wem = 3 nm. Bars represent the fluorescence intensities at 547 nm and each datum was acquired 10 min after HOCl addition.

According to the experimental results and previous reports,37,38,48,49 we speculated that the production of intermediate immonium was firstly induced by Cl+ from the decomposition of HOCl, and the sequent attack of Cl+ resulted in unstable formate ester after the hydrolysis, as a result, 4hydroxy-1,8-naphthalimide was released through the sequent hydrolysis (Scheme 2). Next, the response mechanism of NDMTC for HOCl was further verified. The identification of the hydrolyzates was central to disclose this sensing mechanism because the recognition process of NDMTC for HOCl was very rapid (Fig. S7). Since small molecular weight dimethylmine as a hydrolyzates is difficult to be found in LCMS analysis, O-(N-butyl-1,8-naphthalimide)-4-yl-N,Ndiethylthiocarbamate (NDETC) containing possible hydrolyzate diethylamine was synthesized. As expected, the peaks corresponding to N-butyl-4-hydroxy-1,8-naphthalimide, diethylamine, and sulfinic acid were observed in the high resolving mass spectrum (HRMS) (Fig. S8). Additionally, the control compound O-(N-butyl-1,8-naphthalimide)-4-yl-N,Ndimethylcarbamate (NDMC) could not recognize HOCl even at higher concentration (1.0 mM) (Fig. S9), in which carbamate replaced thiocarbamate. Taken together, these indicate that our proposed recognition mechanism is resonable. Scheme 2. The possible recognition mechanism of fluorescent probe NDMTC for sensing HOCl.

Figure 3. One-photon (a-e) and two-photon (f-j) fluorescence images of live macrophages: (a, f) control macrophages; (b, g) macrophages pretreated with NDMTC (20.0 µM ) for 20 min; (c, h) macrophages pretreated with NDMTC (20.0 µM ) for another 20 min after preincubation with 200.0 μM ABAH for 60 min; (d, i) macrophages pretreated with NDMTC (20.0 µM ) for another 20 min after stimulation with 1.0 μg mL-1 PMA for 60 min; (e, j) relative fluorescence intensities of macrophages in panels a-d and f-i. The provided images of macrophages are representative ones (n = 10 fields of cells). Fluorescence images were obtained with a 405 nm light source (one-photon) and an 800 nm light source (two-photon). Fluorescence emission windows: 470~650 nm. Scale bar is 20 µm.

To test the ability of NDMTC to visualize native HOCl in living systems, we applied NDMTC to live macrophage cell line (RAW 264.7). Prior to the imaging applications, the cytotoxicity of NDMTC was evaluated through 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tests in macrophages. The results reveal that NDMTC has the lower cytotoxicity (Fig. S10a). To improve the accuracy of the intracellular HOCl detection and demonstrate TPM possesses more sensitivity than one-photon microscopy in the bioimaging applications, one-photon and two-photon fluorescence imaging techniques were adopted, repectively. As shown in Fig. 3a and 3f, the weak and negligible background fluorescence was observed from the control macrophages. Interestingly, a bright intracellular fluorescence of macrophages pretreated with NDMTC (20.0 µM) for 20 min was observed (Fig. 3b and 3g), indicating that NDMTC was sensitive enough to detect native HOCl levels in macrophages without external stimulation. Notably, the twophoton imaging of native HOCl with higher resolution was successfully obtained because TPM effectively reduced the background fluorescence by using the long-wavelength excitation. Next, macrophages were preincubated with 4aminobenzoic acid hydrazide (ABAH, a specific inhibitor of

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MPO suppressed the generation of HOCl) and phorbol 12myristate 13-acetate (PMA, a ROS stimulant induced the production of HOCl), respectively. As expected, the weaker fluorescence (pretreatment with ABAH, Fig. 3c and 3h) and stronger fluorescence (pretreatment with PMA, Fig. 3d and 3i) were obtained. These identical results from one-photon and two-photon imaging suggest that NDMTC is capable of monitoring the fluctuation of endogenous HOCl levels in live macrophages, which is of significance for exploring physiological and pathological functions of HOCl. Breast cancer is a serious threat to women health, and its reliable and sensitive diagnosis is a key for the timely remedy. In comparison to normal cells, cancer cells possess higher concentration ROS.50-52 Taking advantages of ultrasensitivity of NDMTC for HOCl, it was used to differentiate between normal and cancerous breast cells by two-photon imaging. As expected, human normal breast cells MCF-10 treated with NDMTC exhibited weak fluorescence, and cancer cells 4T1 showed the stronger intracellular fluorescence, implying that NDMTC was able to differentiate cancerous breast cells from normal cells (Fig. 4). Additionally, the effective distinctions between normal hepatocytes (human normal liver cell line, HL-7702) and hepatoma cells (human hepatoma cell line, HepG2) with the help of NDMTC were also achieved (Fig. S11). So, NDMTC might be helpful for the diagnosis of cancers and other HOCl-related diseases.

ing an alkylmorpholine group to NDMTC framework (Scheme 1). Firstly, the cytotoxicity of Lyso-NDMTC was evaluated through MTT tests in macrophages. The results reveal that Lyso-NDMTC has the lower cytotoxicity (Fig. S10b). To examine the intracellular location of Lyso-NDMTC inside cells, Lyso-NDMTC and Lyso-Tracker Red (a commercially available lysosomal marker) were co-incubated with live HepG2 cells, which was pretreated with NaOCl (10.0 μM) for 30 min. As shown in Fig. 5, the images of green channel and red channel merged well, and it demonstrated that probe LysoNDMTC mainly stained in the lysosome of live cells. Subsequently, the capability of Lyso-NDMTC to visualize lysosomal HOCl levels was performed in live macrophages (Fig. 6). Similar to the investigations on the imaging properties of NDMTC, two-photon fluorescence images of live macrophages pretreated with ABAH and PMA were carried out (Fig. 6). The results suggested that Lyso-NDMTC is capable of monitoring native and the fluctuation of endogenous HOCl levels in lysosomes of live macrophages.

Figure 5. Confocal fluorescence images of live HepG2 cells pretreated with Lyso-NDMTC (20.0 μM ) and Lyso-Tracker Red (100.0 nM ) for 20 min. (a) bright-field image; (b) green channel for probe fluorescence; (c) red channel for Lyso-Tracker fluorescence; (d) merged image of (b) and (c); (e) Intensity correlation plot of Lyso-NDMTC and Lyso-Tracker Red. Scale bar is 10 μm.

Figure 4. Two-photon confocal fluorescence images of live M CF10 and 4T1 cells: (a) control M CF-10 cells; (b) M CF-10 cells pretreated with NDMTC (20.0 µM ) for 20 min; (c) control 4T1 cells; (d) 4T1 cells pretreated with NDMTC (20.0 µM ) for 20 min; (e) relative fluorescence intensities of cells in panels a-d. The provided images of cells are representative ones (n = 10 fields of cells). Fluorescence images were obtained with an 800 nm light source. Fluorescence emission windows: 470~650 nm. Scale bar is 20 µm.

Figure 6. Two-photon confocal fluorescence images of live macrophages: (a) control macrophages; (b) macrophages pretreated with Lyso-NDMTC (20.0 µM) for 20 min; (c) macrophages pretreated with Lyso-NDMTC (20.0 µM ) for another 20 min after preincubation with 200.0 μM ABAH for 60 min; (d) macrophages pretreated with Lyso-NDMTC (20.0 µM ) for another 20 min after stimulation with 1.0 μg mL-1 PM A for 60 min; (e) relative fluorescence intensities of macrophages in panels a-d. The provided images of macrophages are representative ones (n = 10 fields of cells). Fluorescence images were obtained with an 800 nm light source. Fluorescence emission windows: 470~650 nm. Scale bar is 20 µm.

Construction of lysosome-targetable fluorescent probe Lyso-NDMTC for imaging HOCl at the subcellular level. The aforementioned importance of monitoring native HOCl levels in lysosomes motivated us to construct lysosometargetable fluorescent probes for specific determination of HOCl with excellent sensitivity. On the other hand, the pioneering works by Xiao et al demonstrated fully that the alkylmorpholine moiety is an ideal group to target lysosomes.53,54 Encouraged by the excellent HOCl sensing performance of NDMTC, we exploited the corresponding fluorescent probe O-(N-morpholinoethylamino-1,8-naphthalimide)-4yl-N,N-dimethylthiocarbamate (Lyso-NDMTC) by introduc-

Two-photon tissue imaging of HOCl in mice. Inspired by the above exciting results of effective distinction of breast cancer cells from normal cells, we then attempted to

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distinguish cancerous tissues from normal breast tissues in mice. A mouse mammary carcinoma model was constructed by injecting 4T1 cells in forelimb armpit. After 10 days, a tumor mass was obtained. Then, probe NDMTC was injected in normal and tumor tissues, respectively. As shown in Fig. 7, the two-photon imaging of HOCl at the different depths demonstrated that NDMTC possessed good capability of tissue penetration and was capable of detcting HOCl levels at a depth of more than 400 μm in live tissues. Additionlly, the 3D images not only revealed the spatial distribution of HOCl in the breast of mice, but also clearly exhibited tumor tissues have the higher concentration HOCl than normal tissues , which indicated NDMTC could be used to differentiate tumor from normal tissues based on the different concentrations of HOCl (Fig. 7).55 Similar results were also obtained with LysoNDMTC (Fig. S12). Therefore, NDMTC and its lysosometargetable derivative Lyso-NDMTC would provide a promising tool for the visualization of HOCl in vivo.

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for exploring physiological and pathological functions of lower concentration native HOCl in living cells and in vivo.

ASSOCIATED CONTENT Supporting Information. Experimental details and supporting data. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Fax: (86)531 86180017. E-mail: [email protected].

Author Contributions §These authors contributed equally.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by 973 Program (2013CB933800) and National Natural Science Foundation of China (21390411, 21535004, 21227005, 2160070500, and 21107029), Outstanding Young Scientists Award Fund of Shandong Province (BS2013HZ007), and Postdoctoral Science Foundation of China (2013M 541953).

REFERENCES

Figure 7. Two-photon fluorescence imaging at the different depths and the 3D distribution of HOCl in the normal and cancerous breast of mice pretreated with NDMTC (100.0 µM) for 30 min. Fluorescence images were obtained with an 800 nm light source.

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CONCLUSION In conclusion, we firstly designed and synthesized an ICTbased two-photon fluorescent probe NDMTC employing DMTC group as the recognition receptor of HOCl. NDMTC exhibits preeminent selectivity toward HOCl over RNS, biothiols, metal ions, amino acids, and other ROS including OCl-, which might be ascribed to the oxidative cleavage of DMTC moiety via the initiation of an electrophilic addition of Cl+ . Moreover, NDMTC can accurately determine HOCl at the picomolar level and is the first fluorescent probe for the picomolar analysis of HOCl. Experiments on bioimaging applications demonstrate NDMTC can specifically monitor the intracellular native HOCl levels and discriminate cancer cells from normal cells. Subsequently, to track the intrinsic HOCl levels of lysosome in living cells and in vivo, a lysosome-targetable fluorescent probe Lyso-NDMTC was synthesized by introducing an alkylmorpholine group to NDMTC platform, and its ability to image HOCl in the lysosome organelles was further demonstrated. Finally, taking advantages of two-photon deeper tissue penetration, the distinction of tumor tissues from normal tissues in mice was also achieved by the aid of two as-obtained probes. More importantly, this response mechanism would provide a new strategy for constructing specific indicators for HOCl. The present study offers two easy-to-synthesize specific two-photon fluorescent probes for monitoring HOCl at the picomolar level, and an excellent tool

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for TOC only

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Highly Specific and Ultrasensitive Two-Photon Fluorescence Imaging

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