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In Vivo Near-Infrared Fluorescence and Photoacoustic Dual-Modal Imaging of Endogenous Alkaline Phosphatase Xiaoting Gao, Gongcheng Ma, Chao Jiang, Leli Zeng, Shanshan Jiang, Peng Huang, and Jing Lin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00109 • Publication Date (Web): 15 May 2019 Downloaded from http://pubs.acs.org on May 15, 2019

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Analytical Chemistry

In Vivo Near-Infrared Fluorescence and Photoacoustic Dual-modal Imaging of Endogenous Alkaline Phosphatase Xiaoting Gao, Gongcheng Ma, Chao Jiang, Leli Zeng, Shanshan Jiang, Peng Huang, Jing Lin* Marshall Laboratory of Biomedical Engineering, International Cancer Center, Laboratory of Evolutionary Theranostics (LET), School of Biomedical Engineering, Shenzhen University Health Science Center, Shenzhen 518060, China.

ABSTRACT: Alkaline phosphatase (ALP), distributes widely in living organisms, is an important biomarker which closely related to many physiological and pathological processes. However, in vivo real-time detection of ALP remains a big challenge. Herein, we developed a turn-on molecular probe (denoted as LET-3) to visualize ALP activity in tumor tissues through near-infrared fluorescence (NIRF) and photoacoustic (PA) dual-modal Imaging. LET-3, composed of NIR hemicyanine dye (LET-CyOH) and phosphate moiety, showed a 23-fold NIRF enhancement at 730 nm and 27-fold PA enhancement at 710 nm upon activation by ALP, respectively. More importantly, both in vitro and in vivo diagnostic experiments indicated that the LET-3 has a high sensitivity and good selectivity of ALP. These findings provide a promising strategy for in vivo ALP detection by using NIRF and PA dual-channel turn-on probes. Keywords: In vivo, near-infrared fluorescence imaging, photoacoustic imaging, alkaline phosphatase, hemicyanine dye. Alkaline phosphatase (ALP), widely existed in many been employed for in vivo detection.19-22 However, NIRF tissues including liver, bone, intestine, placenta and imaging still suffers from the limited penetration depth kidney, is mainly responsible for the hydrolysis of and low spatial resolution.23,24 Interestingly, phosphate groups in various biological matrices, such as photoacoustic imaging (PAI) could overcome these nucleic acids, proteins and alkaloids.1-4 As a recognized shortcomings. PAI, the combination of optical imaging biomarker, ALP is involved in many critical physiological and ultrasonic imaging, offer real-time, non5,6 and pathological processes. The activity of ALP is invasiveness, deep tissue penetration (up to ≈5 cm) and related to cell differentiation and viability. Furthermore, high spatial resolution.25-37 Therefore, it is worth it was confirmed that abnormal ALP level associated integrating NIRF and PA dual-modal imaging, promising with various diseases such as breast cancer, prostate deep tissue penetration, high sensitivity, and high spatial cancer, heart diseases, bone diseases, diabetes and liver resolution for in vivo imaging and detection.38-43 dysfunction.7-11 Therefore, real-time monitoring of ALP In this study, we developed the first dual-modal activity is of great importance for disease diagnosis. (NIRF/PA) probe (denoted as LET-3) for sensitive and Conventional techniques for ALP detection are mainly selective detection of endogenous ALP. LET-3, a new organic small-molecular compound, was composed of based on electrochemistry,12,13 chromatography,14 surface enhanced resonance Raman scattering NIR hemicyanine dye (LET-CyOH) and phosphate moiety. 15,16 17,18 (SERRS), and colorimetric method. However, In the presence of ALP, phosphate moiety was specifically cleaved, then the LET-3 was converted into these techniques usually not only require complicated LET-CyOH, whose oxygen atom enhanced electronoperations but also could not realize in vivo detection for donating ability,44,45 showed strong intensity biological systems. Recently, near-infrared fluorescence enhancement of both NIRF and PA (Scheme 1). Most (NIRF) imaging, due to its inherent advantages, such as importantly, the as-prepared LET-3 had been real-time, non-invasiveness and high sensitivity, has ACS Paragon Plus Environment

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successfully used for visualizing ALP activity in cancer cells and tumors, which exhibited broad application prospects in pathological research and disease diagnosis.

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anesthesia system (America). PA images were carried out by a Fujifilm Visualsonics Vevo LAZR small animal living photoacoustic imager with the excitation light of 680~970 nm (America). Synthesis. LET-CyOH IR-823 (150 mg, 0.20 mmol) and resorcinol (180 mg, 1.63 mmol) were dissolved in anhydrous dimethylformamide (15 mL) and then anhydrous trimethylamine (0.10 mL, 0.72 mmol) was added. The mixture was stirred at 105 °C for 40 min under a Scheme 1. Schematic illustration of LET-3 for ALP detection in nitrogen atmosphere. After cooling to room temperature, tumor tissues through near-infrared fluorescence (NIRF) and the solvent was removed to give a crude product. After photoacoustic (PA) dual-modal Imaging. purification, LET-CyOH was obtained as a dark blue solid (74 mg, yield: 22.30%). [M+H]+ = 510.10 (calcd for C36H32NO2+ : 509.26). 1H NMR (600 MHz, d6-DMSO) δ EXPERIMENTAL SECTION 8.68 (d, 1H), 8.38 (d, 1H), 8.13 (dd, 2H), 7.84 (d, 1H), 7.74 Chemicals. All chemicals were obtained from Energy-Chemical (dd, 2H), 7.69 – 7.66 (m, 1H), 7.61 (t, 2H), 7.52 (d, 1H), 7.43 – 7.39 (m, 2H), 7.35 (m, 2H), 6.96 – 6.89 (m, 1H), unless otherwise stated. 1,1,26.59 (d, 1H), 5.83 (s, 1H), 4.23 (t, 1H), 4.14 (t, 1H), 3.34 trimethylbenzoindolenine (99%), benzyl bromide (s, 2H), 2.74 – 2.68 (m, 2H), 2.59 (t, 2H), 2.07 (s, 6H). (98%), toluene (99%, Shanghai Lingfeng), ether (99.90%, Shanghai Lingfeng), dimethylformamide LET-3 LET-CyOH (70 mg, 0.14 mmol) was dissolved in (99.80%), tetrahydrofuran (99.50%), phosphorus anhydrous pyridine (10 mL) and then phosphorus oxychloride (0.04 mL, 0.43 mmol) was slowly added. The oxychloride (99.50%), cyclohexanone (99%), absolute mixture was stirred at room temperature for 4 h under a ethanol (99.50%, Macklin), pyridine (99.50%), trimethylamine (99.50%), silica gel (Shanghai Yitan), nitrogen atmosphere, then poured into ice water (200 dichloromethane (99.90%), methanol (99.90%), sodium mL) and stirred overnight. The solvent was removed to orthovanadate (Na3VO4, 99.90%) and trisgive a crude product. After purification LET-3 was obtained as a dark blue viscous oily liquid (53 mg, yield: hydroxymethyl aminomethane (J&K Chemicals, 99.90%). All proteins were obtained from Sigma-Aldrich, America. 40.10%). [M+H]+ = 590.10 (calcd for C36H33NO5P+: 589.23). 1H NMR (600 MHz, CD3OD) δ 8.88 (d, 1H), 8.56 Esterase, catalase, acetylcholinesterase (AChE), thrombin, galactosidase (GAL), bovine serum albumin (dd, 2H), 8.42 (d, 1H), 8.06 (d, 1H), 7.98 – 7.88 (m, 2H), (BSA), trypsin, glucose oxidase (GOx), avidin, lysozyme, 7.80-7.67 (m, 2H), 7.64 – 7.56(m, 1H), 7.54– 7.47 (m, 3H), 7.43 (t, 1H), 7.36 (dd, 2H), 7.20 (dd, 1H), 6.54 (d, 1H), glucose dehydrogenase (GDH), phosphodiesterase 5.74 (s, 1H), 2.81 – 2.71 (m, 2H), 2.65 – 2.51 (m, 2H), 1.96 (PDE), acid phosphatase (ACP) and alkaline phosphatase – 1.83 (m, 2H), 1.41 – 1.27 (m, 6H), 0.97 – 0.83 (m, 2H). (ALP). 13C NMR (150 MHz, CD OD) δ 179.98, 161.37, 153.62, Instrumentation. 3 Mass spectra were measured using Agilent liquid 145.34, 144.45, 139.81, 139.39, 135.82, 133.84, 133.15, chromatography-mass spectrometry (LC-MS) with ESI 132.98, 131.03, 129.88, 129.48, 129.21, 128.88, 128.56, mode (America). Nuclear magnetic resonance (NMR) 128.30, 128.00, 127.89, 126.62, 126.31, 126.10, 122.50, 117.75, 114.79, 111.64, 107.27, 104.02, 52.61, 52.24, spectra were recorded on a Bruker Avance Ⅲ 600MHz superconducting fourier NMR spectrometer (Germany). 31.66, 29.32, 28.91, 26.70, 25.55. UV−vis absorption spectra were obtained on an Agilent Near-infrared Fluorescence and Photoacoustic Technologies Cary 60 UV-vis spectrophotometer Detection for ALP Activity (Malaysia). NIRF spectra were collected on a Themro LET-3 was dispersed in Tris-HCl buffer (50 mM, pH 7.40) Scientific Lumina NIRF spectrometer (America). NIRF to prepare 10 μM and 50 μM solution for NIRF and PA detection of ALP respectively. 0.05 U/mL ALP was added images of cells were acquired on a Leica TCS SP5Ⅱ laser scanning confocal microscope (Germany). NIRF images successively in LET-3 (10 μM) and each dropping was of tumors were performed on a Caliper Life Science IVIS incubated at 37 °C for 2 min. After reacting with 0.00, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, Lumina Ⅱ XGI-8 small animal NIRF imaging and ACS Paragon Plus Environment

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0.55, and 0.60 U/mL ALP, the NIRF intensity was penicillin (100 μg/mL), and streptomycin (100 μg/mL) measured respectively. Similarly, 0.50 U/mL ALP was at 37 °C with 5% CO2. Cells were seeded in 96-well plates added successively in LET-3 (50 μM) and each dropping (5×103 cells/well) and incubated for 24 h. After was incubated at 37 °C for 15 min. After reacting with removing the culture medium, the cells were exposed to 0.00, 0.50, 1.00, 1.50 and 2.00 U/mL ALP, the PA different concentrations of LET-3 (0.00, 0.10, 0.50, 1.00, intensity was measured respectively. In addition, the 2.00, 5.00, 10.00, 20.00, 30.00 and 40.00 μg/mL) for effect of incubation time was also considered. After ALP another 24 h under the same conditions. Then, cell (0.60 U/mL) was added into LET-3 (10 μM), the NIRF viabilities were analyzed by a standard methyl thiazolyl intensity was measured at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, tetrazolium (MTT) assay. To investigate in vivo toxicity 20, 22, 24, 26 and 28 min. Similarly, after ALP (2.0 U/mL) of LET-3, nude mice were injected with Tris-HCl (as the was added into LET-3 (50 μM), the PA intensity was group of control) and LET-3 (5 mg·kg-1), respectively. recorded at 0, 10, 20, 30, 40, 50 and 60 min. For the acute toxicity, a hematoxylin and eosin (H&E) Selectivity of LET-3 to ALP staining analysis of major organs (heart, liver, spleen, lung and kidney) of mice with different treatments was In order to verify the selectivity of LET-3 to ALP, we performed at 24 h post-injection. investigated fourteen proteins, including esterase, catalase, acetylcholinesterase (AChE), thrombin, Cell Imaging galactosidase (GAL), bovine serum albumin (BSA), Intracellular NIRF imaging were conducted according trypsin, glucose oxidase (GOx), avidin, lysozyme, glucose the following procedures. Three groups of HeLa cells dehydrogenase (GDH), phosphodiesterase (PDE), acid were incubated in DMEM supplemented with 10% (v/v) phosphatase (ACP) and alkaline phosphatase (ALP). FBS, penicillin (100 μg/mL) and streptomycin (100 LET-3 was dispersed in Tris-HCl buffer (50 mM, pH 7.40) μg/mL) at 37 °C with 5% CO2. Cells were seeded onto to prepare 10 μM and 50 μM solution for NIRF and PA cover slides at a concentration of 1×105 cells/mL and detection of ALP respectively. For NIRF selectivity cultured in DMEM for 24 h. Then the first group was experiment, concentrations of all proteins were 0.6 replaced with fresh medium containing saline, the U/mL except BSA of 0.6 mg/mL. After incubation with second group was replaced with fresh medium LET-3 (10 μM) at 37 °C for 28 min, NIRF intensities of all containing LET-3 (10 μM), and the third group was proteins were measured. Similar for PA selectivity preincubated with Na3VO4 (500 μM) for 30 minutes experiment, concentrations of all proteins were 2.0 before incubating with LET-3 (10 μM). After incubation U/mL except BSA of 2.0 mg/mL. After incubation with for 1 h, the cells were washed three times with PBS and fixed in a 4% paraformaldehyde for 10 min. After LET-3 (50 μM) at 37 °C for 60 min, PA intensities of all removing the fixative, cells were washed three times proteins were recorded. Moreover, we examined the responsiveness of LET-3 to sodium orthovanadate again with PBS and incubated with diamidino-phenyl(Na3VO4), a recognized ALP inhibitor. For NIRF indole (DAPI) for cellular nuclei staining. At last, the inhibition experiment, ALP (0.6 U/mL) was incubated samples had been prepared for intracellular NIRF imaging. with different concentrations of Na3VO4 (0, 50, 100,150, Tumor Mouse Model 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 and All animal experiments were conducted in accordance 1200 μM) for 30 min, then LET-3 (10 μM) was added for with the Guidelines of the Shenzhen University Animal another 28 min to record NIRF spectra. Similar for PA Care and Use Committee. 5×107 HeLa cells suspended in inhibition experiment, ALP (2.0 U/mL) was incubated 100 μL DMEM which containing 10% (v/v) FBS, with different concentrations of Na3VO4 (0, 50, 100, 150, penicillin (100 μg/mL), and streptomycin (100 μg/mL) 200, 250, 300, 350, 400, 500 and 600 μM) for 30 min, were injected subcutaneously of the mouse to establish then LET-3 (50 μM) was added for another 60 min to tumors in 6-week-old female nude mice. When the collect the PA images. tumor size reached about 60 mm3 (approximately 1~2 Cytotoxicity Assay and Histology Assessment weeks), the nude mice were used for NIRF and PA The cytotoxicity of LET-3 was evaluated on HeLa cells imaging. (Human Cervical Adenocarcinoma Epithelial Cells). HeLa In Vivo Near-infrared Fluorescence and cells obtained from the Chinese Academy of Science Photoacoustic Imaging were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS), ACS Paragon Plus Environment


In vivo NIRF and PA imaging were performed under the same conditions according to the following procedures. Firstly, LET-3 was dissolved in Tris-HCl buffer (50 mM, pH 7.40) to prepare a 500 μM solution. Then, tumorbearing nude mice were divided into three groups and treated differently. Each group contains 5 mice. The first group was untreated as a control group. The second group received an intratumoral injection with LET-3 (500 μM, 50 μL). The third group was intratumorally injected with Na3VO4 (500 μM, 50 μL) and then LET-3 (500 μM, 50 μL). All the NIRF images were obtained with the excitation at 685 nm, and all the PA images were obtained with the excitation at 710 nm. In addition, images at different time points (0, 1, 5, 10, 15, 20, 30 and 40 min) after intratumoral injection with LET-3 (500 μM, 50 μL) in the second group was measured. According to above procedures and conditions, we also intraperitoneally injected LET-3 (500 μM, 50 μL) for NIRF and PA imaging. All the mice were anesthetized with isoflurane (2% in oxygen) during the experiment. RESULTS AND DISCUSSION Probe LET-3 was synthesized via phosphorylation reaction of LET-CyOH with phosphorus oxychloride.46,47 LET-CyOH was obtained by the conjugation of heptamethine cyanine dye IR-823 and resorcinol48. IR823 was synthesized according to the previous reference49 (Figure S1). The chemical structure of LET-3 and intermediate products were characterized by electrospray ionization-mass spectrometry (ESI-MS) and nuclear magnetic resonance (1H NMR, 13C NMR), as shown in the Supporting Information (Figure S2~S9). The photophysical properties of LET-3 were measured in Tris-HCl buffer (50 mM, pH 7.40) at 37 °C. LET-3 exhibited an excellent solubility in the aqueous solution (Figure 1a). The High Resolution Mass Spectra (HRMS) verified the interaction of LET-3 and ALP (Figure S10). Upon the addition of ALP, the absorption band of LET-3 exhibited an obvious red-shift, resulting in a new absorption shoulder between 700~800 nm (Figure 1b). Compared to the initial state of LET-3, the NIRF intensity at 730 nm (NIRF730, λex = 685 nm) and PA intensity at 710 nm (PA710) of the cleaved LET-3 were turned-on with a 23- (Figure 1c) and 27-fold (Figure 1d) enhancement, respectively.

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Figure 1. (a) Color change of LET-3 towards LET-CyOH. (b) UV-vis absorption spectra and (c) NIRF spectra (λex = 685 nm) of LET-3 (10 μM) in the presence or absence of ALP (0.60 U/mL) for 28 min in Tris-HCl buffer (50 mM, pH 7.40) at 37 C. (d) PA spectra of LET3 (50 μM) in the presence or absence of ALP (2.0 U/mL) for 60 min in Tris-HCl buffer (50 mM, pH 7.40) at 37 C.

Next, the sensitivity of LET-3 was investigated based on the ALP concentration and response time. With the addition of ALP (0.00~0.60 U/mL) into the LET-3 solution (10 μM), the absorption intensity of 600~700 nm was decreased gradually while a new absorption shoulder between 700~800 nm was appeared (Figure 2a), and the absorption intensity at 710 nm (Abs710) had a good linear relationship with the concentration of ALP (Figure S11a). Meanwhile, NIRF730 intensity was significantly enhanced (Figure 2b) and linearly correlated with the concentration of ALP (Figure S11b). The detection limit of NIRF imaging was 0.0002 U/mL, which is lower than most of existing fluorescence probes (Table S1). After incubated with different concentrations of ALP (0.00~2.00 U/mL), the PA710 images of LET-3 solutions (50 μM) were obtained (Figure 2c). The results showed that PA710 intensity was proportionally enhanced with the concentration increase of ALP (Figure S11c). The detection limit of PA imaging was 0.8 U/mL. In addition, the reaction kinetics of LET-3 with ALP were also studied. After ALP (0.6 U/mL) was added into the LET-3 solution (10 μM), the Abs710 intensity was gradually increased over time (Figure 2d) and reached a plateau within 28 min (Figure S12a), NIRF730 intensity showed the same varying tendency (Figure 2e and Figure S12b). Similarly, the PA710 intensity of LET-3 solution (50 μM) was gradually increased over time (Figure 2f) and reached a plateau after 60 min (Figure S12c). ACS Paragon Plus Environment

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Figure 2. (a) UV-vis absorption spectra and (b) NIRF spectra (λex

Figure 3. (a) Quantized NIRF730 intensity after LET-3 (10 μM)

= 685 nm) of LET-3 solution (10 μM) upon the addition of ALP

responding to different proteins (0.60 U/mL or 0.60 mg/mL). (b)

(0.00, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55

Quantized PA710 intensity and (c) PA710 images and after LET-3

and 0.60 U/mL) in Tris-HCl buffer (50 mM, pH 7.40) at 37 C. (c)

(50 μM) responding to different proteins (2.0 U/mL or 2.0

PA images (λex = 710 nm) of LET-3 solution (50 μM) upon the

mg/mL). (1: blank, 2: esterase, 3: catalase, 4: AChE, 5: thrombin, 6:

addition of ALP (0.0, 0.5, 1.0, 1.5 and 2.0 U/mL) in Tris-HCl buffer

GAL, 7: BSA, 8: trypsin, 9: GOx, 10: avidin, 11: lysozyme, 12: GDH,

(50 mM, pH 7.40) at 37 C. (d) Abs710 and (e) NIRF730 (λex = 685 nm)

13: PDE, 14: ACP and 15: ALP). (d) NIRF730 intensity of LET-3 (10

intensity of LET-3 solution (10 µM) upon the addition of ALP (0.60

μM) in the presence of ALP (0.60 U/mL) at different Na3VO4

U/mL) in Tris-HCl buffer (50 mM, pH 7.40) at 37 C recorded every

concentrations (0, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800,

2 min. (f) PA710 intensity of LET-3 solution (50 µM) upon the

900, 1000, 1100 and 1200 µM). (e) PA710 intensity of LET-3 (50

addition of ALP (2.0 U/mL) in Tris-HCl buffer (50 mM, pH 7.40) at

μM) in the presence of ALP (2.0 U/mL) at different Na3VO4

37 C recorded every 10 min.

concentrations for 60 min (0, 50, 100, 150, 200, 250, 300, 350, 400, 500 and 600 µM).

The selectivity of LET-3 was also investigated against other proteins including esterase, catalase, acetylcholinesterase (AChE), thrombin, galactosidase (GAL), bovine serum albumin (BSA), trypsin, glucose oxidase (GOx), avidin, lysozyme, glucose dehydrogenase (GDH), phosphodiesterase (PDE) and acid phosphatase (ACP). NIRF730 intensity showed turn-on performance in the presence of ALP (Figure 3a), while other proteins could not induce any significant changes (Figure S13). As we know, the proteins mentioned above themselves have no PA signals (Figure S14). When they were incubated with ALP, their PA710 intensity didn’t increase except ALP (Figure 3b), indicating LET-3 had good selectivity for ALP (Figure 3c). Furthermore, the LET-3 could be used for the screening of ALP inhibitors. Sodium orthovanadate (Na3VO4) was tested as a phospholipase inhibitor. The results showed the ALP activity decreased with concentration increase of Na3VO4, leading to the weakening of NIRF730 (Figure 3d) and PA710 (Figure 3e) intensities. When the concentrations of Na3VO4 were 1200 μM and 600 μM, the inhibition on NIRF (Figure S15a) and PA (Figure S15b) detection were maximized, respectively.

Figure 4. Confocal microscope fluorescence images of HeLa cells. From top to bottom: untreated control, cells treated with LET-3 solution (10 μM) only, and cells treated with Na3VO4 (500 μM) and LET-3 (10 μM).

The cytotoxicity and biocompatibility of LET-3 were evaluated on HeLa cells by using standard methyl thiazolyl tetrazolium (MTT) assay. The cell viability remained at a high level after 24 h incubation with different concentrations of LET-3 solution (0~40 μM), suggesting no obvious cytotoxicity of LET-3 (Figure S16). Subsequently, NIRF imaging was conducted on the cell level to validate the capability of LET-3 for ALP detection. Due to the intracellular over-expressed ALP, HeLa Cells incubated with LET-3 solution (10 μM) exhibited strong ACS Paragon Plus Environment


fluorescence signal. On the contrary, cells pre-treated with Na3VO4 (500 μM) displayed no fluorescence signal, due to the inhibition of ALP activity by Na3VO4 (Figure 4). These results indicated that the LET-3 could monitor the ALP activity in living cells. We also investigated in vivo toxicity of LET-3. Compared with the control group, there was no noticeable organ damage or inflammation of the group treated with LET3, suggesting good biocompatibility of LET-3 (Figure S17). In addition, we measured fluorescence signals of major organs of normal healthy mice to determine the bio-distribution of LET-3, suggesting LET-3 was mainly distributed in the liver and spleen after intravenous injection (Figure S18). The feasibility of LET-3 was used as a NIRF/PA dualmodal imaging probe to monitor ALP activity in the subcutaneous HeLa xenograft model. After intratumoral injection of LET-3 (500 μM, 50 μL), the NIRF730 intensity was gradually enhanced (Figure 5a) and reached its maxima at 40 min (Figure 5b). While, the control group and the group pretreated with Na3VO4 (500 μM, 50 μL) were observed negligible NIRF730 signals (Figure 5c), compared with 15.06-fold enhancement of the group only treated with LET-3 (Figure 5d). Correspondingly, under the same procedures and conditions, NIRF730 signals of the mice injected intraperitoneally with LET-3 (500 μM, 50μL) also changed significantly (Figure S19).

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As shown in Table S1, the existing probes for in vivo ALP detection are only used as fluorescence imaging probes, however, LET-3 was also employed for PA detection of ALP in tumor to realize the double checking. As expected, mice treated with LET-3 showed obvious PA710 signal which increased with time (Figure 6a). After 40 min, PA710 intensity was strongest (Figure 6b). PA710 signals of the control group and the group pre-injected with Na3VO4 (500 μM, 50 μL) had not been observed (Figure 6c). Compared with the control group, LET-3 induced 9.75-fold enhancement in tumor-bearing mice (Figure 6d). Similarly, PA opening ability of the probe was also confirmed in mice intraperitoneally injected with LET-3 (Figure S20). In a word, all these results suggested that LET-3 could efficiently monitor ALP activity in vivo through NIRF and PA imaging.

Figure 6. (a) Representative PA710 images of tumor-bearing nude mice that received an intratumoral injection of LET-3 (500 μM, 50 μL) after incubation for 0, 1, 5, 10, 15, 20, 30 and 40min. (b) Relative PA710 intensity of (a). (c) Representative PA710 images of the group of control, the group received an intratumoral injection of Na3VO4 (500 μM, 50 μL) and then LET-3 (500 μM, 50 μL), the group only received an intratumoral injection of LET-3 (500 μM, 50 μL). (d) The corresponding PA710 amplitude of mice with different treatments.

CONCLUSIONS In summary, we developed the first dual-modal turn-on molecule probe (LET-3) for NIRF and PA imaging of ALP. Figure 5. (a) Representative NIRF730 images (λex = 685 nm) of LET-3 can be specifically activated by ALP, triggering the tumor-bearing nude mice that received an intratumoral injection significantly NIRF and PA signals enhancement. With its of LET-3 (500 μM, 50 μL) after incubation for 0, 1, 5, 10, 15, 20, 30 excellent biocompatibility, LET-3 was successfully and 40 min. (b) Relative NIRF730 intensity of (a). (c) employed in living tumor-bearing mice for real-time Representative NIRF730 images (λex = 685 nm) of the group of monitoring of ALP. Especially, this study constructed the control, the group received an intratumoral injection of Na3VO4 dual-modal imaging probe to detect endogenous ALP in (500 μM, 50 μL) and then LET-3 (500 μM, 50 μL), the group only living organisms which breaking the barrier of singlereceived an intratumoral injection LET-3 (500 μM, 50 μL). (d) The modal imaging probes. Therefore, LET-3 has great corresponding NIRF730 intensity changes of mice toward different potential for early diagnosis of ALP-related diseases, treatments. such as cervical carcinoma, chondropathy, cirrhosis, liver cancer and breast cancer. ACS Paragon Plus Environment

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Analytical Chemistry (11) Ooi, K.; Shiraki, K.; Morishita, Y.; Nobori, T. J. Clin. Lab. Anal.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Additional information as noted in the text (Figures S1S20); Synthesis, characteristic (1H NMR, 13C NMR and ESI-MS spectra), UV-Vis absorption, fluorescence and photoacoustic spectra.

2010, 21, 133-139. (12) Ino, K.; Kanno, Y.; Arai, T.; Inoue, K. Y.; Takahashi, Y.; Shiku, H.; Matsue, T. Anal. Chem. 2012, 84, 7593-7598. (13) Goggins, S.; Naz, C.; Marsh, B. J.; Frost, C. G. Chem. Commun. 2014, 51, 561-564. (14) Lakra, S.; Jadhav, V. J.; Garg, S. R. Food. Anal. Method. 2016, 9, 2002-2009. (15) Ruan, C.; Wang, W.; Gu, B. Anal. Chem. 2006, 78, 3379-3384. (16) Ingram, A.; Moore, B. D.; Graham, D. Bioorg. Med. Chem. Lett.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected] Notes The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is financially supported by the startup fund from the Shenzhen University (2017090), the National Natural Science Foundation of China (31771036, 51703132), the Basic Research Program of Shenzhen (JCYJ20180507182413022, JCYJ20170412111100742), the Guangdong Province Natural Science Foundation of Major Basic Research and Cultivation Project (2018B030308003), the Fok Ying-Tong Education Foundation for Young Teachers in the Higher Education Institutions of China (161032). We thank Instrumental Analysis Center of Shenzhen University (Xili Campus).

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