Near-Infrared Fluorescent Probes for Hypoxia Detection via Joint

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Near-Infrared Fluorescent Probes for Hypoxia Detection via Joint Regulated Enzymes: Design, Synthesis, and Application in Living Cells and Mice Xinwei Tian, Zhao Li, Yue Sun, Pan Wang, and Huimin Ma Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04249 • Publication Date (Web): 30 Oct 2018 Downloaded from http://pubs.acs.org on October 30, 2018

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Analytical Chemistry

Near-Infrared Fluorescent Probes for Hypoxia Detection via Joint Regulated Enzymes: Design, Synthesis, and Application in Living Cells and Mice Xinwei Tiana, Zhao Lia,*, Yue Sunb, Pan Wangb and Huimin Mac,* a Shaanxi

Engineering Laboratory for Food Green Processing and Safety Control, College

of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi'an 710062, China b

Ministry of Education Key Laboratory of Medicinal Resources and Natural

Pharmaceutical Chemistry, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi’an 710062, China c

Beijing National Laboratory for Molecular Sciences Key Laboratory of Analytical

Chemistry for Living Biosystems Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China * Corresponding author (E-mail: [email protected]; [email protected])

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ABSTRACT Hypoxia detection is emphasized with attention due to tumor and related diseases diagnosis, which could provide useful methods for exploring the mechanism of hypoxic tumor. Herein, we report two unprecedented hypoxia-sensitive probes that specifically switch-on their near-infrared fluorescence signals in the presence of hypoxia up-regulated enzymes (nitroreductase and cytochrome P450 reductase). The probes were designed by featuring the decomposition of IR-780 coupled to hypoxia activatable p-nitrobenzyl or azo moiety, which exhibit near-infrared fluorescence emission, high sensitivity, selectivity, stable photo-stability, and low cytotoxicity. Besides, the joint use of two probes could differentiate the 4T1 and HepG2 cells lines through fluorescence signals successfully. More importantly, applied to monitor hypoxia in 4T1 tumor-bearing BALB/c mice, the two probes have ideal biodistribution with passive accumulation and fast clearance, and there is negligible organ damage by hematoxylin and eosin staining analysis. To the best of our knowledge, there is no fluorescent probe for hypoxia detection via joint hypoxia regulated enzymes reported so far. This method may be of great potential use in cancer and other relevant diseases diagnosis.

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INTRODUCTION Hypoxia is defined as a decrease oxygen level within the tissue and perceived as a typical feature of many diseases, such as inflammatory diseases1,2, stroke3, cardiac ischemia4 and solid tumors5,6. In the condition of hypoxia, tumor cells can be stimulated and secrete vascular growth factors to promote the formation of abnormal blood vessels, and then the tumor will metastasize and deteriorate7,8. In addition, the hypoxic microenvironment has a screening effect on tumor cells, and increases the degree of tumor malignancy, and then, tumor cells have resistance to chemotherapy or radiotherapy9. Thus, the development of efficient methods for detecting hypoxic conditions in tumor cells and in vivo is strongly desirable. Hypoxia-sensitive fluorescent probes can be used to monitor the hypoxic status of tumor cells via the detection of endogenous up-regulated reductase, which have attracted much attention due to high sensitivity and high spatial and temporal resolution10-12. Detection of up-regulated level of nitroreductase (NTR) and cytochrome P450 reductase (CYP450 reductase) within hypoxic tumor tissue using fluorescence probe methods has been described recently, as reviewed by Qian, Li, and Ma et al. They reported several fluorescence probes bearing aromatic nitro groups to visible-excited fluorophores (such as cyanine dyes, resorufin and nile blue) for NTR detection13-16. Freeman and Nagano et al. reported that probes modified azo group are sensitive to hypoxia17-19. Besides, near-infrared (NIR) fluorescent probes with superior properties such as excellent tissue

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penetration, low biological and autofluorescence damage are developted for hypoxic tumor imaging20,21. Not confined in vitro, research working in tandem with establishment of models related to hypoxia diseases have been developed22-25. Nevertheless, all the works are based on the detection of single enzyme in tumor, which lose diagnostic comparative value of joint reductase detection. Research on cell hypoxia by monitoring both intracellular NTR and CYP450 reductase would be helpful to better reveal the properties of cancer cells. To the best of our knowledge, there is no fluorescent probe for hypoxia detection via joint hypoxia regulated enzymes reported so far. In

this

work,

two

near-infrared

hypoxia-sensitive

probes

(E)-3,3-dimethyl-2-(2-(6-((4-nitrobenzyloxy)carbonyla-mino)-2,3-dihydro-1H-xanthen-4 -yl)vinyl)-1-propyl-3H-indolium (AXNO2) and 2-((E)-2-(6-((E)-(4-(dimethylamino)phenyl)diazenyl)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3,3-dimethyl-1-propyl-3H-indo lium (AXNN) are presented for imaging the hypoxic status of tumor cells and mice via their reactions with the hypoxia up-regulated enzymes (nitroreductase and cytochrome P450 reductase). The probes were designed by introducing hypoxia activatable p-nitrobenzyl or azo moiety as the sensing unit to the hemicyanines with an amino group (AXPI), synthesized through two-step decomposition of IR-780, exhibiting superior analytical performances such as high stability, tunable optical properties and NIR fluorescence emission26. Reaction of the two probes with hypoxia up-regulated enzymes would lead to the specially appointed enzymatic cleavage or reduction and thus the release of fluorophore AXPI27-30. Ideally each hypoxic probe should only react with NTR or CYP450 reductase to cause corresponding fluorescence ‘turn-on’, reduction on each ‘reaction dot’ to release fluorophore AXPI. Such fluorescence responses lead to the

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establishment of a highly sensitive and selective method for monitoring hypoxia up-regulated enzymes activity, as well as for imaging the hypoxic status of tumor cells and mice in vivo.

Scheme 1: A) Synthesis of probe AXNO2 and AXNN. Reagents and conditions: (i) 3-Nitrophenol, K2CO3, CH3CN, rt, 4 h; (ii) SnCl2, HCl, CH3OH, 70 ℃ , overnight; (iii) triphosgene, 4-Nitrobenzyl alcohol, CH3CN, rt, 3 h; (iv) TFA, NaNO2, sulfamic acid, N, N-dimethylaniline, DMF, MeCN/CH2Cl2 (1:4), rt, 2 h. B) The proposed activation mechanisms of probe AXNO2 with Nitroreductase and AXNN with Cytochrome P450 Reductase.

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NO2

Cl

NH2

O

NI

N

N

(i)

O N

I-

NI

(ii)

AXPI

N

(iii)

(iv)

O HN

O

N

N

NO2 O

O N

A

N I-

I-

AXNN

AXNO2 ne

B

1e

O2 O2

O HN

N

O N H

X

N

N

O N

O I-

N I-

X=O or H

3e

NH

+ CO2

NH2

NH2 N O NI

AXPI

EXPERIMENTAL SECTION Apparatus. NMR spectra were acquired on a Brucker DMX-600 spectrometer in CD3OD, using tetramethylsilane (TMS) as an internal standard. Electrospray ionization mass spectrum (ESI-MS) was taken on Shimadzu LC-MS 2010A instrument (Kyoto, 6


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Japan). A Hitachi U-3010 spectrophotometer was used to measure the UV-vis absorption spectra, and fluorescence spectra were obtained by HITACHI F-7000 Fluorescence Spectrometer (Hitachi Limited Ltd, Japan) with a Xenon lamp and 1-cm path length quartz cuvettes at the slits of 10/10 nm. MTT analysis was recorded on a microplate reader (BIO-TEK Synergy HT, USA). Fluorescence imaging of cells was conducted using a confocal laser scanning microscope (Leica, Germany) with an excitation wavelength at 635 nm and mice imaging was performed on a Bruker in vivo xtreme imaging system. Reagents. Nitroreductase from Escherichia coli, Cytochrome P450 Reductase from Rabbit liver, nicotinamide adenine dinucleotide phosphate (NADPH) and IR-780 iodide were

purchased

from

Sigma-Aldrich

Co.

Ltd.

3-Nitrophenol

(99%),

N,N-Diisopropylethylamine (99.5%) and 4-Nitrobenzyl alcohol (99%) were purchased from Acros Organics Co. Ltd. Triphosgene from Adamas Reagent Co. Ltd. A phosphate buffered saline solution was obtained from Invitrogen Company. Stock solution (1 mM) of AXNO2 and AXNN were prepared by dissolving a refined calculation of probe in deoxygenated dimethyl sulphoxide (DMSO). Thin layer chromatography (TLC) analysis was performed on silica gel and the silica gel (200-300 mesh) was used for the column chromatography. Reactive oxygen species (ROS) including NO2ˉ, ClOˉ, ·OH and H2O2 were prepared. 5-6 week old female BALB/c mice were obtained from the Experimental Animal Center of the Fourth Military Medical University (Xi’an, China). All other chemicals used were of analytical grade without purification. Synthesis of fluorophore AXPI. Compound AXPI was synthesized according to our previous reported method26.

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Synthesis of probe AXNO2. To a stirred solution of AXPI (45 mg, 0.11 mmol) in CH3CN (5 mL) and DIPEA (60 mg, 0.50 mmol) was added a mixture of triphosgene (80 mg, 0.26 mmol) in CH3CN dropwise. The mixture was allowed to react for 2 h in ice bath, then the ice bath was removed, and heated to reflux over a period of 3 h. The reaction solution was cooled at room temperature and diluted with dichloromethane (10 mL), filtered and 4-Nitrobenzyl alcohol (46 mg, 0.3 mmol) was added. The mixture was reacted at room temperature for an additional three hours, monitored by TLC. The solvent was purified via flash chromatography on a silica column (CH2Cl2/MeOH as an eluent) to afford compound AXNO2 as a mazarine solid (23.5 mg, 0.4 mmol, 36.4%). 1H NMR (600 MHz, CD3OD) δ 8.60 (d, J = 14.9 Hz, 1H), 8.17 (d, J = 8.7 Hz, 2H), 7.71 (s, 1H), 7.63 (d, J = 7.3 Hz, 1H), 7.59 (d, J = 8.6 Hz, 2H), 7.55-7.40 (m, 4H), 7.37 (d, J = 8.4 Hz, 1H), 7.28-7.23 (m, 2H), 6.46 (d, J = 14.9 Hz, 1H), 5.23 (s, 2H), 4.30 (s, 2H), 2.72 (d, J = 5.6 Hz, 2H), 2.64 (d, J = 5.8 Hz, 2H), 1.91 (dd, J = 14.2, 7.0 Hz, 4H), 1.78 (s, 6H), 1.06 (t, J = 7.4 Hz, 3H).13C NMR (150 MHz, CD3OD) δ 179.33 (s), 162.57 (s), 154.99 (s), 154.65 (s), 148.91 (s), 146.96 (s), 145.34 (s), 144.14 (s), 143.42 (s), 142.96 (s), 134.50 (s), 130.31 (s), 129.52 (s), 129.42 (s), 129.17 (s), 128.57 (s), 124.68 (s), 123.86 (s), 118.57 (s), 117.07 (s), 115.76 (s), 114.12 (s), 66.48 (s), 52.09 (s), 47.77 (s), 30.12 (s), 28.49 (s), 25.03 (s), 22.36 (s), 21.57 (s), 11.69 (s). ESI-MS, m/z calcd. for AXNO2 (C36H36N3O5+, [M]+): 590.2649; found: 590.2640 (Figure S1-S3). Synthesis of probe AXNN. To a solution of AXPI (45 mg, 0.11 mmol) in MeCN/CH2Cl2 (1 : 4, 5 mL) containing 1% TFA solution were mixed, and the mixture was stirred at 0 °C under an argon atmosphere. Subsequently, NaNO2 (15 mg, 0.22 mmol) was added to the mixture. The reaction mixture was allowed to stir at the same

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temperature for 30 min, and then sulfamic acid (20 mg, 0.2 mmol) was added and stirring for 5 min. Immediately, N, N-dimethylaniline (92 µL, 0.72 mmol) in MeCN (1 mL) was added and stirred for 2 h, and then the crude product was diluted with dichloromethane. The residue was purified via flash chromatography on a silica column (CH2Cl2/MeOH as an eluent) to afford AXNN as a deep green solid (30.7 mg, 0.057 mmol, 51.6%). 1H NMR (600 MHz, CD3OD) δ 8.24 (d, J = 15.0 Hz, 1H), 7.69-7.63 (m, 1H), 7.59 (dd, J = 8.0, 1.4 Hz, 1H), 7.52 (d, J = 8.8 Hz, 2H), 7.46-7.42 (m, 2H), 7.39 (d, J = 8.1 Hz, 1H), 7.34 (s, 1H), 7.21 (s, 1H), 7.07 (s, 1H), 6.54 (d, J = 8.9 Hz, 2H), 6.06 (d, J = 15.0 Hz, 1H), 3.97 (t, J = 7.6 Hz, 2H), 3.06 (s, 6H), 2.65-2.50 (m, 2H), 2.30 (s, 2H), 1.82-1.69 (m, 10H), 0.98 (t, J = 7.3 Hz, 3H).13C NMR (150 MHz, CD3OD) δ 179.27 (s), 161.04 (s), 155.79 (s), 154.76 (s), 154.46 (s), 146.66 (s), 144.21 (s), 143.43 (s), 142.58 (s), 132.91 (s), 131.72 (s), 130.28 (s), 129.44 (s), 128.94 (s), 126.78 (s), 124.00 (s), 123.88 (s), 121.88 (s), 116.14 (s), 114.31 (s), 112.63 (s), 108.12 (s), 106.20 (s), 52.21 (s), 47.97 (s), 40.43 (s), 30.22 (s), 28.38 (s), 24.65 (s), 22.28 (s), 21.17 (s), 11.62 (s). ESI-MS, m/z calcd. for AXNN (C36H39N4O+, [M]+): 543.3118; found: 543.3116 (Figure S4-S6). General procedure for NTR and CYP450 reductase assay. AXNO2 and AXNN were used at a final concentration of 10 μM. Absorption and fluorescence spectra of AXNO2 with NTR enzymatic reactions were performed at 37 °C in a 2 mL total volume of PBS buffer (10 mM, pH 7.4) in the presence of 100 μM NADPH. For CYP450 reductase assay, argon gas was bubbled into solution to create the hypoxic environment, the corresponding spectra of AXNN with CYP450 reductase enzymatic reactions were performed at 37 °C in a 2 mL total volume of PBS buffer (10 mM, pH 7.4, 1% DMSO) in the presence of 100 μM NADPH. For comparison, the solution containing no NTR or

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CYP450 reductase (control) was measured under the same conditions at the same time. Cytotoxicity Assay. Similar to the procedures reported in literatures31, the cytotoxicity of probe to HepG2 and AT1 cells were conducted by the standard MTT assay. Fluorescence imaging in Cells. 4T1 cells were cultured in high glucose DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. HepG 2 cells were cultured in RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were cultured for 12 h under various oxygen conditions (20% O2, 10% O2, 5% O2 and 1% O2) before fluorescence imaging, then the cells were incubated with 10 μM probe for 30 min and subsequently rinsed with PBS buffer to remove the free probe. The pixel intensity using Image J software [(version 1.37c, National Institutes of Health (NIH)] was measured from at least 10 cells. Fluorescence imaging in 4T1 breast cancer model. 4T1 breast cancer model preparation procedures were in accordance with the guidelines of the Institutional Animal Care and Use Committee. Female BALB/c mice weighing 20-25 g at the age of 5-6 week were used in this study 0.1 mL 4T1 cells suspension containing approximately 4×105 4T1 cells was orthotopically injected into the right quadrant of the abdomen of mice. The size of the tumor was measured using a Vernier caliper. Mice with tumor size approximately 7-10 mm in the greater diameter, reached on the 12th-14th day after inoculation, were used for in vivo measurements. For in vivo imaging, probe AXNO2 or AXNN (500 µM, in 100 µL PBS) were injected into tumor bearing mice through tail vein and fluorescence images were taken by Bruker in vivo xtreme imaging system after injection at 0.5, 4, 12 and 24 h. A filter set

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(λex = 650 nm, λem = 700 nm) was used for the measurement. The mice were kept on the imaging stage under anesthetized condition with isoflurane gas in oxygen flow during the imaging process. After injection at 24 h, tumors and other organs (heart, liver, spleen, lung, kidney) were excised and imaged under the same system as mentioned above. Histological Studies. Similar to the procedures reported in literatures32. The mice were sacrificed after 14 days of treatment. The heart, liver, spleen, lung and kidney were fixed in 4% paraformaldehyde, and then paraffin embedded sectioning was conducted for hematoxylin and eosin (H&E) staining under standard protocols, and observation by optical microscope. Animal preparation procedures were in accordance with the guidelines of Experimental Animal Administration published by the State Committee of Science and Technology of People’s Republic of China.

RESULTS AND DISCUSSION Probe AXNO2 and AXNN were synthesized and characterized using 1H NMR, 13C NMR and MS. The detailed synthetic steps and characterization of probe AXNO2 and AXNN are given in the Supporting Information (Figures S1-S6 and Scheme 1). Then we proceeded to collect the absorption and fluorescence spectra of probes toward NTR or CYP450 reductase in PBS (pH 7.4, 10 mM, 1% DMSO) in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. In PBS, probe AXNO2 and AXNN displayed an absorption maxima at around 570 nm and 450 nm, respectively. Treatment of 10 µM AXNO2 with 10 µg/mL NTR or 10 µM AXNN with 8 µg/mL CYP450 reductase resulted in a decrease of respective characteristic absorption peak,

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concomitant with an additional shoulder peak at around 670 nm (Figure 1A, 1C). Fluorescence intensity of probe AXNO2 and AXNN themselves were weakly as the efficient strong withdrawing ability of nitro groups and -N=N- bond33,34. Reaction of both two probes with substrate produce a fluorescence response at 706 nm in virtue of probes were established by incorporating different recognition moiety into the same stable hemicyanine skeleton AXPI (Figure 1B, 1D). Moreover, the long analytical wavelength feature (λex/em=670/706 nm) of the reaction system is favorable for in vivo imaging studies35-37.

Figure 1. Spectral profiles of 10 μM AXNO2 and AXNN incubation with NTR and CYP450 reductase, respectively. (A) Absorption spectra of 10 μM AXNO2 before (a) and after (b) reaction with 10 μg/mL NTR; (B) Fluorescence spectra (λex = 670 nm) of 10 μM AXNO2 reacting with NTR at different concentrations (0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8 and 12


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10 μg/mL). (C) Absorption spectra of 10 μM AXNN before (a) and after (b) reaction with 8 μg/mL CYP450 reductase; (D) Fluorescence spectra (λex = 670 nm) of 10 μM AXNN reacting with CYP450 reductase at different concentrations (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6 and 8 μg/mL). The reaction was performed in PBS (pH 7.4, 10 mM, 1% DMSO) at 37 °C in the presence of 100 μM NADPH and collected until reach stable fluorescence intensity. λex/em = 670/706 nm. We then measured the kinetic parameters of probe AXNO2 and AXNN by adding of a known concentration reductases. As depicted in Figure S7 in the Supporting Information, the fluorescence increase could reach a peak value within 8 min of AXNO2 assay. By contrast, no significant fluorescence change was observed in AXNO2 without NTR (control) within the same period of time, which indicates that AXNO2 is highly stable in the detection system. In the case of AXNN, the fluorescence intensity could reach a peak value within 30 min, which is known to be a CYP450 reductase sensitive probe with short fluorescence saturation time38-41. Next, we choose the simulated physiological conditions as the reaction of probe AXNO2 with NTR (reaction at 37 °C for 8 min in 10 mM PBS of pH 7.4), and AXNN with CYP450 reductase (reaction at 37 °C for 30 min in 10 mM PBS of pH 7.4) in the presence of 100 µM NADPH. The curve was plotted with the fluorescence intensity at 706 nm, the fluorescence response of AXNO2 to NTR at varied concentrations is shown in Figure 1B, and a good linear equation of y = 390.3980x (μg/mL) + 195.5304 (R2 =0.9952) was obtained between the fluorescence intensity and the NTR concentration in the range of 0.5-4.0 μg/mL, the detection limit is determined to be 53 ng/mL NTR. The confirmation mechanism of CYP450 reductase sensitive probe was demonstrated using 13



CYP450 reductase placed under hypoxic conditions. There is a good linear equation of y = 201.4760x (μg/mL) +29.0531 (R2 =0.9939) was obtained in the range of 0.5-4.0 μg/mL (Figure 1D), the detection limit is determined to be 22 ng/mL CYP450 reductase. For further evaluation of sensing selectivity of the system for the reductases, the probes were investigated by treating with various potential interfering species. As depicted in Figure S8 and Figure S9 in the Supporting Information, the fluorescence of two probes could not be triggered on in the presence of a panel of inorganic salts or reactive oxygen species, including MgCl2, CaCl2, ZnSO4, FeCl3, NO2ˉ, ClOˉ, ·OH, and H2O2, common species as glucose, lactose, arginine, serine and glusate. In addition, some reducing species and enzymes including hydrogen sulfide (H2S), ascorbic acid (Vc), FeCl2, glutathione transferase (GST), DT-diaphorase (DTD), and carboxylesterase (CaE) were also investigated. It is satisfactory that significant activation occurred only in the presence of NTR or CYP450 reductase, and there is no interference between two enzymes detection. The results collectively indicated the high specificity of the two probes. To confirm the mechanism shown in Scheme 1, the reaction products of both probes were analyzed by spectroscopic test and ESI-MS analysis. The spectroscopic properties of fluorophore AXPI were investigated, revealing that the absorption and emission spectra of NTR and CYP450 reductase catalyzed reaction solution are in accordance with AXPI, implying the production of AXPI (Figure S10). More importantly, the ESI-MS analysis further certified that the NTR and CYP450 reductase metabolite is AXPI (m/z 411.2[M]+; Figure S11 and Figure S12 in the Supporting Information). These observations show that the reaction of AXNO2 with NTR in the presence of NADPH

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would result in the reduction of the p-nitrobenzyl moiety, accompanied by spontaneously collapses to release fluorophore AXPI. For CYP450 reductase assay, the efficient "OFF-ON" switch of fluorescence was realized by the incursion of azo moiety (-N=N-), which reduced by CYP450 reductase to regenerate the original fluorophore AXPI. These results indicated that both probes have the function of diagnosis cancer via hypoxia up-regulated enzymes, with selective identifying hypoxic cells and tumor. Cytotoxicity is an important indicator of probe feasibility in biological system, we then tested the potential toxicity of AXNO2 and AXNN to cells by standard MTT assay31 (Figure S13). The results showed that there was no significant change in cell viability, indicating the well-tolerated by cells. Considering the good biocompatibility and detection range in cuvette, we chose probe AXNO2 and AXNN at 10 μM to conduct the following cells study. In addition, the skillfully-convictively application in cancer cells is particularly noteworthy and we anticipated that the probes have the capability for monitoring endogenous NTR and CYP450 reductase in living cells. As shown in Figure S14, both HepG 2 and 4T1 cells themselves display no fluorescence (column a) under the excitation of 635 nm, however, if the cells pretreated under various oxygen conditions (20% O2, 10% O2, 5% O2 and 1% O2), and then treated with probe AXNO2, the fluorescence in cells would increase with the decrease of O2 level. The pixel intensity of the cells was analyzed using Image J (version 1.37c, NIH), the fluorescence intensity from 4T1 and HepG2 cells incubated under different hypoxic (10%, 5% and 1% O2) conditions increases by ca.1.02, 1.39, and 2.82 times for 4T1 cells, 0.75, 2.52, and 4.27 times for HepG2 cells, respectively, with respect to that incubated 4T1 cells under normoxic

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conditions. In case of AXNN, the pixel intensity was also obtained by the above calculation method, 4T1 and HepG2 cells incubated under different hypoxic increases by ca.1.16, 1.77, and 3.55 times for 4T1 cells, 1.21, 2.81, and 4.89 times for HepG2 cells, respectively; with respect to that incubated 4T1 cells under normoxic (Figure S15 for CYP450 reductase assay). The preceding results indicate that probe AXNO2 and AXNN can be used to visualize the hypoxic status of a wide variety of tumor cell lines.

Figure 2. (A) Co-culture of 4T1 and HepG2 cells: (a) cells induced by 1% O2 for 12 h; (b) cells induced by 1% O2 for 12 h, and then incubated with AXNO2 (10 μM) for 30 min; (c) cells induced by 1% O2 for 12 h, and then incubated with AXNN (10 μM) for 30 min; (d) cells induced by 1% O2 for 12 h, then incubated with AXNO2 (10 μM) and AXNN (10 μM) for 30 min. The red arrow points to HepG2 cells. (B) Relative pixel intensity (n = 10) of the corresponding fluorescence images. [the pixel intensity of 4T1 cells from the image b is defined as 1.0]. Cancer cells related reductases has been seen as biomarkers for differentiation between normal cells and tumor cells as they only expressed in hypoxic cancer cells42-44. Of greater significance, the expression value of these biomarkers overlap provide the potential value to differentiate cancer cells lines. In our experiments, 4T1 cells (5×105/mL) and HepG2 (1×106/mL) cells were plated on 20 mm glass bottom dish and 16


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allowed to adhere. After that, the cells were grown under hypoxic conditions of 1% O2 for 12 h and then incubated with the probe AXNO2 or AXNN. According the pixel intensity analyse for AXNO2, the fluorescence intensity of HepG2 cells in this condition is about 1.37-fold higher than that of 4T1 cells (Figure 2A, column a). In contrast, the fluorescence intensity of HepG2 cells under hypoxic conditions of 1% O2 is about 1.28-fold higher than that of 4T1 cells (Figure 2A, column b). To increase the fluorescence contrast of the two cell lines, the cells were first incubated with 10 µM AXNO2 and 10 µM AXNN together. The results are satisfactory, the fluorescence intensity of HepG2 cells in this condition is about 1.60-fold higher than that of 4T1 cells by the pixel intensity analysis (Figure 2A, column c). To the best of our knowledge, our study represents the first example of joint detection of hypoxia up-regulated enzymes to differentiate two kinds of cancer cells. In the therapeutic diagnosis of breast cancer (BC), molecular biomarkers have been introduced for aiding tumour classification or improving prediction of response to specific antitumor agents. Currently, as specific tumor markers for BC, only including oestrogen,

progesterone,

human

epidermal

growth

factor

receptor

2

and

receptorshormone receptors are used in routine clinical practice45-47. Despite new markers were constantly discovered, most of them are tested in vitro, which lost the spatial-temporal controllability. NTR and CYP450 reductase were expected to become new evaluation standards of tumor. On the basis of the excellent performance of probe AXNO2 and AXNN, here we make such an attempt. In our experiments, probe AXNO2 and AXNN were intravenously (i.v.) injected into 4T1 tumor-bearing BALB/c mice for in vivo imaging of hypoxia tumor.

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The changes of fluorescence intensity in tumor were monitored at time point of 0.5, 4, 12, and 24 h after injection. As shown in Figure 3A, obvious fluorescence signals were obtained from tumors, clearly distinguishable from neighboring tissues. The quantitative results revealed that 12 h post-injection was the optimal time point for tumor hypoxia region in order to obtain the maximum efficacy (Figure 3B). Furthermore, the downward trend of fluorescence image between 12 and 24 h was analyzed and noted that both the two probes had fast uptake and clearance kinetics. To determine that the fluorescence signal was from the probe accumulated in the tumour region, the mice were sacrificed after injection at 24 h and major organs (heart, liver, spleen, lung, kidney, and tumor) were captured (Figure 3C). As only specific reduction in tumor region, the results are consistent with the in vivo observation. Finally, the toxicity of probes were assessed, we evaluated the five tissues (heart, liver, spleen, lung, kidney) by hematoxylin and eosin (H&E) after 14 th day. There was negligible organ damage (no necrosis, edema, inflammatory infiltration, or hyperplasia) in the sections of the seven organs, indicating that at the dose used here, AXNO2/AXNN showed good biosafety. The results into the fluorescence imaging via joint hypoxia regulated enzymes detection have led to an increase in our knowledge of endogenous up-regulated standards in tumor. Furthermore, using our new synthesized NIR fluorescence probes as visual indicators, combined with their biological safety and spatial orientation ability, can improve the diagnostic sensitivity of malignant cancer, and it is beneficial for early diagnosis, treatment and prognosis evaluation of cancer.

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Figure 3. (A) Time-dependent in vivo fluorescence imaging of BALB/c mice after tail vein injection of AXNO2 and AXNN (100 µL, 500 µM). (B) Quantification analysis of average tumor fluorescence intensity after injection for different periods of time (0, 0.5, 4, 12 and 24 h). (C) Fluorescence images of major organs (heart, liver, spleen, lung, kidney and tumor) after 24 h injection. (D) H&E staining images of the major organs (heart, liver, spleen, lung and kidney) treated with saline, AXNO2 or AXNN. The organs for H&E staining were collected after 14 days of treatment.

CONCLUSIONS In conclusion, we have reported the design, preparation and application of two probes (AXNO2 and AXNN), which could be specifically reduced by hypoxia up-regulated enzymes in tumor for fluorescence-guided imaging. Notably, such a detection has been first used in differentiate HepG2 and 4T1 cells through fluorescence signals. Importantly, both probes have the ideal biodistribution with passive accumulation 19



and fast clearance, the safety was further evidenced by H&E staining analysis. The results indicate that a set of biological markers, rather than a single one, seem to be more reliable for prognostic tumor hypoxia. This method may be of great potential use in cancer and other relevant diseases diagnosis.

ASSOCIATED CONTENT Supporting Information Additional information, as noted in the text. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected]. ORCID Zhao Li: 0000-0001-7702-3348; Huimin Ma: 0000-0001-6155-9076 ACKNOWLEDGMENT 20


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We are grateful to the financial support from the National Natural Science Foundation of China (Nos.21605099, 21820102007 and 21621062), the Fundamental Research Funds for the Central Universities, China (GK201802019 and 2018CSLZ007), and Shaanxi Province Agricultural Science and Technology Innovation and Key Project (2018NY-099). Financial support from the Young Talent Fund of University Association for Science and Technology in Shaanxi, China (20180207) is also greatly appreciated.

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For TOC only

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Near-Infrared Fluorescent Probes for Hypoxia Detection via Joint

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