Targeted Myocardial Hypoxia Imaging Using a Nitroreductase

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Targeted Myocardial Hypoxia Imaging Using a NitroreductaseActivatable Near-Infrared Fluorescent Nanoprobe Yunshi Fan, Mi Lu, Xie-an Yu, Miaoling He, Yu Zhang, Xiaonan Ma, Junping Kou, Bo-Yang Yu, and Jiangwei Tian Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00298 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 17, 2019

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Analytical Chemistry

Targeted Myocardial Hypoxia Imaging Using a NitroreductaseActivatable Near-Infrared Fluorescent Nanoprobe Yunshi Fan‡, Mi Lu‡, Xie-an Yu, Miaoling He, Yu Zhang, Xiao-Nan Ma, Junping Kou, Bo-Yang Yu*, and Jiangwei Tian* State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Research Center for Traceability and Standardization of TCMs, Cellular and Molecular Biology Center, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, P.R. China ABSTRACT: Development of a highly selective and sensitive imaging probe for accurate detection of myocardial hypoxia will be helpful to estimate the degree of ischemia and subsequently guide personalized treatment. However, an efficient optical approach for hypoxia monitoring in myocardial ischemia is still lacking. In this work, a cardiomyocyte-specific and nitroreductase-activatable nearinfrared nanoprobe has been developed for selective and sensitive imaging of myocardial hypoxia. The nanoprobe is a liposome-based nanoarchitecture which is functionalized with a peptide (GGGGDRVYIHPF) for targeting heart cells and encapsulated a nitrobenzene-substituted BODIPY for nitroreductase imaging. The nanoprobe can specifically recognize and bind to angiotensin II type 1 receptor that is overexpressed on the ischemic heart cells by the peptide and is subsequently uptaken into heart cells, in which the probe is released and activated by hypoxia-related nitroreductase to produce fluorescence emission at 713 nm. The in vitro response of the nanoprobe towards nitroreductase resulted in 55-fold fluorescence enhancement with the limit of detection as low as 7.08 ng/mL. Confocal fluorescence imaging confirmed the successful uptake of nanoprobe by hypoxic heart cells and intracellular detection of nitroreductase. More significantly, in vivo imaging of hypoxia in a murine model of myocardial ischemia was achieved by the nanoprobe with high sensitivity and good biocompatibility. Therefore, this work presents a new tool for targeted detection of myocardial hypoxia and will promote the investigation of the hypoxia-related physiological and pathological process of ischemic heart disease.

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Myocardial ischemic injury is one of the major public health problems and a major cause of morbidity and mortality worldwide.1,2 This disease means insufficient blood and disproportion between the amount of oxygen and substrates supplied to the heart and the amount needed by the heart, leading to anaerobic metabolism and reduced contractile function.3-5 Myocardial hypoxia, refers to decreased coronary blood flow caused by various conditions including myocardial infarction and coronary disease, can result in inadequate supply of myocardial oxygen and reduced metabolite clearance.6,7 Because of a high coronary arteriovenous difference, the myocardium cannot bring about a substantial improvement in oxygen supply by the increased extraction of oxygen from the blood. Therefore, myocardial hypoxia is a significant physiological and pathological feature of myocardial ischemia, and the development of diagnostic agent for accurate detection of the hypoxic level will be helpful to estimate the degree of ischemia and subsequently guide personalized treatment. Hypoxia is accompanied by elevated levels of reductive enzymes especially nitroreductase (NTR).8-10 Under hypoxic conditions, NTR can effectively catalyze the reduction of nitroaromatic substrates to the corresponding amines in the presence of nicotinamide adenine dinucleotide (NADH) as a

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source of reducing equivalents.11 The nitro groups in substrates can be reduced to a nitroso group, then a hydroxylamine group, and ultimately an amino group.12 According to the previous reports, NTR-catalyzed reactions have been utilized in design of positron emission tomographic (PET) imaging probes for myocardial hypoxia diagnosis.13-15 For example, nitroimidazole compound-based contrast agents have been widely used for PET imaging of myocardial hypoxia.16 The principle is that when the compound enters into the hypoxic cell, the nitro group is reduced to be amino group by intracellular NTR. The amines can remain trapped within the cell and be detected using PET imaging.17 However, the signal-to-noise ratio is low and the radioactive materials are harmful to the body. Alternatively, fluorescence imaging provides another promising approach because it offers the advantages of simplicity, high sensitivity, noninvasive measuring, and safety.18-20 Hence, development of NTR-activatable fluorescent probe for myocardial hypoxia imaging is highly desired. In light of high fluorescence quantum yield, excellent photochemical stability, high extinction coefficient and low biological toxicity,21-23 boron dipyrromethene (BODIPY) dye had been selected as the scaffold to design the NTR-activatable probe in this work. The 3, 5-positions of the BODIPY were

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substituted with styryl to gain extended π conjugation system, which makes maximum emission peak redshift to increase the penetrability of light and minimize the interference of autofluorescence from body.24,25 Furthermore, 2-nitrobenzene as a quenching and recognizing moiety was introduced to the BODIPY skeleton to obtain the probe (BDP-NO2) for NTR detection. Under normal conditions, the fluorescence of BDPNO2 was off with a fluorescence quantum yield (ΦF) of 0.001, which ensured a low background signal. Upon reaction with NTR under hypoxia, it qualified a near-infrared (NIR) emission centered at 713 nm. Due to the degradability and biocompatibility,26 liposome is used as the nanocarrier to encapsulate BDP-NO2. Taking the phenomenon that the amino acid sequence of Gly-Gly-Gly-Gly-Asp-Arg-Val-Tyr-Ile-HisPro-Phe (GGGGDRVYIHPF) can target to angiotensin II type 1 (AT1) receptor that is overexpressed on the ischemic heart cells into account,27-29 a diagnostic nanoprobe (Pep/BDPNO2@Lip) is designed with BDP-NO2 encapsulation and the peptide modification (Scheme 1). After the occurrence of myocardial ischemia, the blood vessels in the left ventricle became leaky, which promoted the penetration of nanoprobe in a manner similar to enhanced permeability and retention effect.30 The nanoprobe could specifically recognize myocardial cells via selective binding between GGGGDRVYIHPF and AT1 receptor, and be selectively taken up into myocardial cells. In that case, BDP-NO2 was released from the nanoprobe with a “turn-on” fluorescent signal appearing owing to that BDP-NO2 was reduced to BDP-NH2 by NTR. Furthermore, the newly designed nanoprobe had been successfully used for fluorescence imaging of hypoxic cardiomyocytes and real-time monitoring of ischemia in the heart. Scheme 1. Schematic Illustration of Structure and Function of Pep/BDP-NO2@Lip for NTR-Activatable Imaging of IschemiaInduced Myocardial Hypoxia

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EXPERIMENTAL SECTION Synthesis of BDP-NO2. BDP-NO2 was synthesized as shown in Scheme 2. A solution of 1,3,5,7-tetramethyl-meso-(2nitrophenyl)-BODIPY (100 mg, 0.27 mmol), benzaldehyde (175 mg, 0.75 mmol) and catalytic amount of p-TsOH in a mixture of toluene (50 mL) and piperidine (1 mL) was placed in a round bottom flask equipped with a Dean Stark trap, the mixture was heated at its boiling point until it had evaporated to produce a dry residue. The resulting solid was dissolved in dichloromethane and washed with water three times. The organic phase was dried over MgSO4 and the solvent was evaporated under reduced pressure, and the resulting crude residue was purified by silica-gel flash column chromatography (25% ethyl acetate/hexane) and recrystallized from CH2Cl2/Hexane to provide the desired compound as dark red solid (61 mg, yield 29%). 1H NMR (400 MHz, CDCl3): δ 8.19 (m, 1 H), 7.83-7.70 (m, 5 H), 7.65-7.63 (m, 4 H), 8.19 (m, 1 H), 7.44-7.39 (m, 4 H), 7.35-7.29 (m, 3 H), 6.66 (s, 2 H), 1.44 (s, 6 H). 13C NMR (100 MHz, CDCl3) δ: 153.25, 148.89, 140.94, 136.91, 136.62, 134.11, 133.41, 132.85, 131.66, 130.72, 130.38, 129.21, 128.93, 127.76, 125.05, 119.32, 118.38, 14.20. HRMSESI: m/z: calcd [M+Na]+, m/z= 568.1978, found m/z = 568.1980 (Figures S1−S3). Scheme 2. Synthetic Routine of BDP-NO2

Synthesis of Pep/BDP-NO2@Lip. Pep/BDP-NO2@Lip was prepared by the thin-film hydration method as previously described.31 Briefly, a mixture of dioleoyl phosphoethanolamine (DOPE), cholesterylhemisuccinate (CHEMS), cholesterol and DSPE-PEG2000-GGGGDRVYIHPF at a molar ratio of 6:4:3:0.5 was dissolved in 3 ml of chloroform: methanol (2:1 v/v). 2 mL chromatogram class acetonitrile containing 0.8 mg BDP-NO2 was added to the above solution and mixed well to obtain a mixture solution. Then the solution was evaporated to dryness under vacuum using a rotary evaporator (Shanghai Yarong Biochemistry Instrument Company, China) in a 50 mL roundbottom flask at 55 °C for about 20 min, forming the thin lipid film on the bottom. The film was then flushed with nitrogen gas for 1 h and vacuum dried overnight to remove any traces of residual solvent. Subsequently, the dried lipid film was suspended and hydrated in 10 mL of sterile PBS buffer (pH 7.4) at 55 °C and kept hand-shaking until the lipid film was taken off the flask wall. The suspension was sonicated using an ultrasonics processor (KUDOS, China) in an ice bath for 15 min to reduce vesicle size and was further downsized by extrusion 10 times through a 0.22 μm polycarbonate membrane. Using 106 MW cutoff cellulose ester tubing (Spectra/Por Biotech, Ranco Domingues, CA) to remove nonencapsulated dye by dialysis against PBS. The GGGGDRVYIHPF modified and Sulfo-


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1 Cyanine5 carboxylic acid (Cy5) loaded liposome 58 2 (Pep/Cy5@Lip) was prepared according to the similar method 59 3 described above. For the preparation of nontargeted liposomes 60 4 containing BDP-NO2 (BDP-NO2@Lip) or Cy5 (Cy5@Lip), 61 5 similar procedures were carried out except that the equivalent 62 6 molar DSPE-PEG2000-GGGGDRVYIHPF was replaced by 63 7 DSPE-PEG2000. 64 8 Confocal Fluorescence Imaging for Living Cells. Rat heart 65 9 myocardium H9c2 cells were seeded into 35-mm confocal 66 10 dishes (Glass Bottom Dish) at a density of 1 × 104 per dish and 67 11 incubated at 37 °C under normoxic or hypoxic conditions for 68 12 different periods of time. The medium was then replaced with 69 13 fresh serum-free culture medium containing Pep/BDP- 70 14 NO2@Lip (10 μg/mL) and incubated at 37 °C for different 71 15 times under the respective conditions. Cell imaging was then 72 16 carried out after washing cells three times with PBS buffer (pH 73 17 7.4) to remove any liposomes on the surface of cells. The 74 18 fluorescence of cells was visualized with a confocal laser 75 19 scanning microscope with a 63 × oil immersion objective lens at 76 20 stationary parameters including the laser intensity and exposure 77 21 time. The fluorescence signal of cells incubated with Pep/BDP- 78 22 NO2@Lip was excited with a 633 nm helium-neon laser and 79 80 23 emission was collected from 685 to 735 nm. 24 For the colocalization assay of the Pep/BDP-NO2@Lip- 81 25 loaded H9c2 cells, the cells were washed with PBS and further 82 26 incubated with 100 nM LysoTracker Green or 100 nM 83 27 MitoTracker Green for 15 min. LysoTracker Green and 84 28 MitoTracker Green were excited at 488 nm with an argon ion 85 86 29 laser and the emission was collected from 505 to 535 nm. 30 Fluorescence in Vivo Imaging and Myocardial Injury 87 31 Biomarkers Detection. The left anterior descending coronary 88 32 artery was ligated to induce ischemia. After 6 h of ligation, the 89 33 mice were intravenously injected with 100 μL 0.4 mg/mL 90 34 Pep/BDP-NO2@Lip or BDP-NO2@Lip at a dose of 2 mg/kg via 91 35 tail vein. The in vivo fluorescence imaging was then performed 92 36 to detect fluorescence signal. At 8, 9, 10 and 12 h post ligation, 93 37 mice were sacrificed and then their chests were opened for 94 38 cardiac imaging respectively. Afterwards, the heart of the mice 95 39 as well as other major organs were harvested, and subjected for 96 40 ex vivo imaging and semiquantitative analyses. All images were 97 41 analyzed by Maestro 3.0 software. After imaging, the hearts of 98 42 mice were harvested to examine the histopathology by H&E 99 43 staining. 100 44 Myocardial ischemic injury was assessed by measuring 101 45 Creatine Kinase (CK), Creatine Kinase Isozyme (CK-MB), 102 46 Cardiac Troponin I (cTnI), and Myoglobin (Mb) levels. At 12 103 47 h after ligation, blood samples were collected and centrifuged at 104 48 3500 rpm for 15 min at 4 °C to separate serum. The levels of CK, 105 49 CK-MB, cTnI, and Mb were measured using relevant assay kits. 106 107 50 108 51 RESULTS AND DISCUSSION 109 52 Optical Properties of BDP-NO2. The spectroscopic 110 53 evaluation of BDP-NO2 before and after reacting with NTR in 111 54 phosphate buffer (pH =7.4, 10 mM) at 37 °C were investigated. 112 55 BDP-NO2 exhibited a very weak absorption in the long- 113 56 wavelength region. After reacting with NTR, it produced a 114 57 relatively strong absorption at about 675 nm (Figure S4A). As 115

shown in fluorescence emission scan, the BDP-NO2 showed nearly non-fluorescent with a fluorescence quantum yield (Φf) 0.001 (Figure S4B), which was attributed to the strong fluorescence quenching of the nitro group. However, the reaction of BDP-NO2 with NTR triggered a significant fluorescence enhancement at 713 nm. A 55-fold increase in its fluorescence intensity indicated that BDP-NO2 was considerably sensitive for the detection of NTR. Optical Response of BDP-NO2 to NTR. The kinetics of the fluorescence intensity between the BDP-NO2 and NTR was further investigated by the fluorescence emission technique. After the addition of 1 μg/mL NTR, the fluorescence emission scan showed a dramatic enhancement and no significant increase was observed after 30 min (Figure S5A). Fluorescence kinetic curves of the BDP-NO2 reacting with different concentrations of NTR were detected. The results revealed that higher concentrations of enzyme accelerated reduction of the nitro-containing BDP-NO2 to the fluorescent amine and resulted in stronger fluorescence intensity (Figure S5B). Moreover, the fluorescence increase could reach the plateau in around 30 min with NTR of no more than 10 μg/mL. On the other hand, the fluorescence signal of BDP-NO2 without NTR remained almost unchanged during the same period of time, which indicated that the BDP-NO2 was sufficiently stable in the reaction system. The reduction of BDP-NO2 and Pep/BDP-NO2@Lip by NTR were optimized in terms of pH, temperature and concentration of NADH, respectively. The results revealed that the changes of pH value from 4 to 10, temperature from 20 to 40 °C hardly influenced the fluorescence of the BDP-NO2. However, the turn-on signal could be obtained after the reaction with NTR. For the BDP-NO2, it functioned well at pH 7.4 and 37 °C, and the fluorescence intensity reached its maximum at a NADH concentration of 200 μM (Figures S6). Due to the acid sensitivity of liposomes, the BDP-NO2 could be released under acidic conditions. Strong fluorescence of the reaction system was observed at about pH 6.0 (Figures S7), at which value NTR still had a strong catalytic activity. Previous research discovered that the intracellular pH will decrease at a pH of around 6.0 during the ischemia process.32,33 Thus, the reaction of Pep/BDP-NO2@Lip with NTR could proceed effectively under myocardial ischemia conditions. Under the optimized conditions (reaction at 37 °C for 30 min in 10 mM PBS of pH 7.4 in the presence of 200 μM NADH), the fluorescence response of BDP-NO2 was increased with increasing concentrations of NTR (Figure 1A). Besides, the increase showed a good linearity with the NTR concentration in the range of 0.1-1.0 μg/mL (Figure 1B). The detection limit (3S/k, in which S is the standard deviation of blank measurements, n = 12, and k is the slope of the linear equation) was determined to be 7.08 ng/mL NTR, which was lower than most of the existing fluorescent probes for NTR detection.34-36 In addition, kinetic parameters for the enzyme-activated reaction of BDP-NO2 were investigated under the optimized condition. The Lineweaver–Burk plot of 1/V (V is the initial reaction rate) versus 1/concentration was established (Figure S8). By fitting the data with the Michaelis–Menten equation, the calculated apparent Michaelis constant Km and the



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44 45 46 47 48 49 50 To know the chemoselectivity of BDP-NO2 toward NTR, 51 various biologically relevant species such as salts (K+, Na+, Ca2+, 52 Mg2+), amino acids (cysteine, arginine), biothiols (glutathione, 53 dithiothreitol), DT-diaphorase, glucose, vitamin C, vitamin B6, 54 reactive oxygen species (H2O2, HClO), and human serum 55 albumin (HSA) were tested in parallel under the same 56 conditions (Figure S10). We observed that all of the above 57 potentially interfering species show no interference toward the 58 reaction between BDP-NO2 and NTR, suggesting the high 59 60 selectivity of BDP-NO2 for NTR detection. 61 An enzyme inhibition test was carried out to demonstrate that 62 the turn-on signal of BDP-NO2 to NTR arose from the enzyme63 catalyzed reduction. Dicoumarin as a common NTR activity 64 37 inhibitor was utilized (Figure S11). Compared with the group 65 without dicoumarin (curve C), fluorescence intensity was much 66 lower when BDP-NO2 was pre-treated with 0.1 mM dicoumarin 67 (curve D). Higher concentration of dicoumarin (0.2 mM) led to 68 a further decrease in fluorescence intensity of BDP-NO2 (curve 69 E), illustrating that the NTR activity can be inhibited by 70 dicoumarin. Moreover, enzyme inhibition was performed at 71 4 °C and no significant fluorescence response was observed 72 (curve F). These results confirmed that the fluorescence off-on 73 response of the BDP-NO2 to NTR is attributed to the enzyme74 promoted reductase reaction. 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 Figure 1. (A) Fluorescence response of 5 μM BDP-NO2 in the 91 presence of 200 μM NADH to NTR at varied concentrations (0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, 6.0, 8.0 and 10 μg/mL). (B) 92 Relationship between the concentration of NTR and the 93 fluorescence intensity of the reaction mixture. All measurements 94 were acquired at 37 °C in 10 mM PBS, pH 7.4, λex/em = 640/713 nm. 95 (C) TEM image of Pep/BDP-NO2@Lip negatively stained with 2.0% 96 (w/v) phosphotungstic acid. Scale bars: 200 nm. (D) In vitro 97 release profiles of BDP-NO2 from Pep/BDP-NO2@Lip at different 98 pH conditions. Data are means ± SD (n=3). 99 100

maximum rate Vmax were 44.17 μM and 0.071 μm s-1, respectively, indicating strong affinity between the enzyme and the BDPNO2. To confirm the sensing mechanism, the reaction of BDPNO2 with NTR was conducted using ESI-MS analysis (Figure S9). The ESI-MS spectrum of the reaction solution of BDP-NO2 with NTR showed a major peak at m/z = 538.2228 [M+Na+], which was characterized as BDP-NH2.

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Characterization of Pep/BDP-NO2@Lip. BDP-NO2 was loaded and distributed in the liposome phospholipid bilayer, and the targeting peptide was modified on the surface. Transmission electron microscopy (TEM) image showed welldispersed spherical structure of the nanoprobe (Figure 1C). The average hydrodynamic diameter was measured to be 133 nm by dynamic light scattering (DLS) (Table S2). The nanoprobe remained stable for at least half a month in both PBS and culture media with negligible increase in particle size (Figure S12A). In addition, the size is small enough to accumulate in the ischemic area via EPR effect.38 To calculate the encapsulation efficiency (EE) and loading content (LC) of the nanoprobe, the standard curve of BDP-NO2 was obtained (Figure S13). The EE of BDP-NO2 in BDP-NO2@Lip and Pep/BDP-NO2@Lip were measured to be 89.6 ± 0.13% and 84.3 ± 0.54%, respectively (Table S1). The LC of BDP-NO2 in BDP-NO2@Lip and Pep/BDP-NO2@Lip were measured to be 16.2 ± 0.08% and 11.4 ± 0.06%, respectively. Compared with that of BDP-NO2@Lip, zeta potential of Pep/BDP-NO2@Lip was shifted from -35.46 mV to -28.72 mV (Table S2), which was due to the change in surface charge upon modification with the peptide containing basic amino acids. In vitro release of the BDP-NO2 from the nanoprobe was evaluated in different pH buffer mediums. The release rate of BDP-NO2 was much faster at acidic environment and reached a plateau in about 6 h (Figure 1D). The cumulative release of BDP-NO2 at pH 7.4 was only 29%, whereas it increased to be 76% at pH 6.0 (Figure S12B). Therefore, it was reasonable to assume that liposomal nanovehicle allow pH-controlled BDP-NO2 release. To investigate the stability of the nanoprobe in physiological condition, the release of the BDP-NO2 from Pep/BDPNO2@Lip was evaluated in mouse serum. It indicated that BDPNO2 released slowly from the nanoprobe in serum, and the accumulative release percentages was only 23.4% within 48 h (Figure S14). Because the release of BDP-NO2 was only controlled by acidic pH, the nanoprobe was stable and the leakage was negligible in physiological condition. Fluorescence Imaging of NTR in Hypoxic Cells. Since BDPNO2 is off-state, an always-on fluorescent reagent Cy5 (λex = 649 nm; λem = 670 nm) was loaded into the liposome to fabricate the Pep/Cy5@Lip nanoprobe in order to investigate the binding specificity of GGGGDRVYIHPF to AT1 receptor on hypoxic cardiomyocytes. For the H9c2 cells incubated with Pep/Cy5@Lip under normoxia or Cy5@Lip under hypoxia, the Cy5 fluorescence was very weak. In contrast, the fluorescence increased significantly for the hypoxic H9c2 incubated with Pep/Cy5@Lip (Figure S15A), indicating an enhanced cellular uptake by the peptide under hypoxic condition. L-02, HK-2 and HUVEC cells were also incubated with Pep/Cy5@Lip to perform fluorescence imaging. There was almost no fluorescence observed in these cells (Figure S15B). The flow cytometric results further demonstrated the targeting ability to hypoxic cardiac cells by the peptide (Figure 2). The biocompatibility of BDP-NO2 and Pep/BDP-NO2@Lip was investigated under normoxic and hypoxic conditions, respectively. In the presence of BDP-NO2 at 1-40 μM or nanoprobe at 0.5-50 μg/mL, the cellular viabilities were more


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46 47 The intracellular uptake of Pep/BDP-NO2@Lip was 48 investigated in H9c2 cells using confocal laser scanning 49 microscopy. Firstly, the cells were cultured under hypoxic 50 conditions with 2% O2. Then, 10 μg/mL of nanoprobe was 51 added to H9c2 cells at specific time and incubated for a total of 52 10 h under hypoxic conditions, respectively. After incubation, 53 the red fluorescence signal was observed in the cells, which 54 increased gradually along with the incubation time and reached 55 a maximum at 4 h. With the increase of the incubation time to 8 56 h, the fluorescence intensity was hardly changed (Figure S17). 57 Therefore, 4 h was selected as the optimal incubation time for 58 the nanoprobe. It indicated the efficient uptake and subsequent 59 60 activation of the nanoprobe by hypoxia-induced reduction. Next, Pep/BDP-NO2@Lip was used to image the hypoxic 61 status by monitoring NTR in H9c2 cells. H9c2 cells were 62 incubated with Pep/BDP-NO2@Lip and grown under 63 normoxic (20% O2) for 16 h or hypoxic (2% O2) conditions for 64 different times (2, 4, 6, 10 and 16 h) at 37 °C. Cells treated with 65 nanoprobe under normoxic conditions showed almost no 66 67 excited 68 69 70 71 72 73 74 75 76 77 78 79 80 81 Figure 2. Flow cytometric assay of H9c2, L-02, HK-2 and HUVEC 82 cells targeting in vitro. 83 fluorescence emission, whereas a notable time-dependent 84 increase in fluorescence signals was observed within 10 hours in 85 hypoxic cells (Figure 3). To quantitatively compare the changes 86 in fluorescence intensity, mean pixel fluorescence intensity 87 analysis was made by using ImageJ software (National Institutes 88 of Health, USA), averaging at least five H9c2 cells. Under 2 h 89 and 10 h hypoxic conditions, the fluorescence intensity 90 increased by about 4.5 times and 14.4 times respectively, 91 compared with normoxia (Figure S18A). This result suggested 92 that the BDP-NO2 could be efficiently released from Pep/BDP- 93 NO2@Lip and subsequently catalytically reduced under 94 hypoxia, thus viable for in vitro imaging of hypoxic cardiac cells. 95 Additionally, when the hypoxia time was extended to 16 h, the 96 fluorescence intensity did not increase significantly compared 97 with 10 h, which may be due to the decreased activity of NTR 98 resulted from apoptosis. Flow cytometry provided quantitative 99 results that were consistent with the qualitative results (Figure 100 S18B). In addition, the content of NTR and total protein 101 concentration was quantified to investigate the expression level 102 than 80% after incubation for 12 h (Figure S16), indicating good biocompatibility of the nanoprobe.

of NTR in cells under normoxia and hypoxia (Figure S19). The

concentration was defined as NTR content expressed per unit of protein. Compared with that in normoxic cells, the level of NTR in hypoxic cells elevated gradually along with the increaing time of hypoxia, further demonstrating the feasibility of NTR as a hypoxic trigger. In order to study the subcellular location of released BDPNO2 in cells, the nanoprobe-incubated H9c2 cells were costained with LysoTracker Green and MitoTracker Green (commercial lysosomal and mitochondrial trackers that have no fluorescence response to NTR) under hypoxic conditions, respectively. Incubation of 10 μg/mL Pep/BDP-NO2@Lip with the hypoxic cells (2% O2 for 10 h) for 4 h was followed by addition of 100 nM LysoTracker Green or MitoTracker Green (15 min incubation). The results clearly showed the appearance of a red signal characteristic of the reduction of BDP-NO2 by NTR. The green channel illustrated visualization of the lysosome or mitochondria. Most of the red fluorescence overlapped with the green MitoTracker fluorescence, and the changes in the intensity profiles of the linear region of interest (ROI) across the cells were synchronous in the two channels (Figure S20). On the other hand, the red signal did not match what was observed in the case of LysoTracker Green (Figure S21). This result indicated that the reaction between BDP-NO2 and intracellular NTR was basically completed in mitochondria. Notably, some red fluorescent spots from BDP-NH2 that did not overlapped with the green fluorescence of MitoTracker were also observed in the cells. The reason was that long-time of hypoxia could induce mitochondrial membrane permeabilization (MMP),39,40 which led to the diffusion of BDPNH2 from mitochondria to cytoplasm. We also adjusted the hypoxia time of the cells from 10 h to 5 h. Co-localization results showed that the red fluorescence superimposed well with the green MitoTracker fluorescence (Figure S22), further demonstrating NTR activatable imaging in mitochondria and MMP-induced BDP-NH2 diffusion. To further demonstrate that the fluorescence in cells loaded with nanoprobe was caused by the catalytic reduction of NTR, dicoumarin as a reductase inhibitor was utilized again to decrease NTR activity. The fluorescence from H9c2 cells that were pretreated with dicoumarin was significantly weaker than that the cells without dicoumarin (Figure S23), illustrating that the NTR activity can be largely inhibited by dicoumarin in the cells. Further verification by flow cytometry yielded the same experimental results. This inhibition confirmed that the fluorescence increase of BDP-NO2 in the hypoxic H9c2 cells is caused by the function of endogenous NTR and that the Pep/BDP-NO2@Lip can be used to image the change of NTR. Chemically induced cell hypoxia imaging was evaluated by using cobalt chloride (CoCl2) to stimulate H9c2 cells to elevate the intracellular NTR level.41,42 It is found that the cytotoxic effect of CoCl2 on H9c2 cells may be related to its induction of oxidative stress, leading to the loss of balance between the oxidation and the antioxidation in H9c2 cells and eventually to the oxidative stress-induced injury cells.43 As presented, after the treatment of CoCl2, a remarkably stronger fluorescence intensity in H9c2 cells was observed than that detected under normal conditions (Figure S24). Therefore, the results revealed



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that intracellular NTR production was effectively promoted under the administration of CoCl2. Besides, as is shown

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Figure 3. Confocal fluorescence images and differential interference contrast (DIC) images of H9c2 cells under normoxic (20% O2) for 16 h and hypoxic (2% O2) conditions for different times (2, 4, 6, 10, 16 h). H9c2 cells cultured under different conditions were incubated with 10 μg/mL Pep/BDPNO2@Lip. Scale bars: 20 μm.

in the figure, the strong fluorescence signal could be attenuated effectively by pretreating with N-acetyl cysteine (NAC), which remediated cell injury via the consumption of ROS levels by increasing the GSH level, offering obvious protective effect on H9c2 cardiomyocytes against injuries induced by chemical hypoxia.44 The data confirmed that the nanoprobe was capable of monitoring dynamic changes in NTR level at cellular concentrations and visualizing the cell hypoxia caused by CoCl2. Flow cytometry analysis was conducted to further confirm the different fluorescence response of Pep/BDP-NO2@Lip by measuring the intensity of red fluorescence in H9c2 cells (Figure S24). The consequence was consistent with the fluorescence microscopy images shown above.

In Vivo Fluorescence Imaging of NTR in Myocardial Ischemic Mouse Model by Pep/BDP-NO2@Lip. We next examined whether Pep/BDP-NO2@Lip can detect hypoxia in the whole animal in vivo using a myocardial ischemic mouse model. Myocardial ischemia was induced by ligation of the left

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anterior descending coronary artery as described.45 As for sham group, the artery was not ligated. After 6 h of ligation, Pep/BDPNO2@Lip with a dose of 2 mg/kg was administered to mice by intravenous injection via tail vein. After 8 h post ligation, a significant enhancement in fluorescence intensity was showed in the heart region. As the ligation time was extended, the fluorescence signal further enhanced and the heart could be distinguished from the surrounding normal tissue (Figure 4A). After 12 h, the highest fluorescence intensity (ca. 11-fold higher) was attained (Figure S25A), demonstrating the targeted delivery, hypoxia-activatable fluorescence of Pep/BDPNO2@Lip in ischemic heart. The time-dependent fluorescence signal appeared at the heart site after ligation, revealing that most of the Pep/BDP-NO2@Lip was effectively taken up by cardiomyocytes and the released BDP-NO2 generated amino product under catalytic reduction of NTR induced by hypoxia. As a control, a same dose of Pep/BDP-NO2@Lip was injected into the sham group mice (Figure 4A). The fluorescence in heart was faintly visible and the fluorescence intensity was not increased over time (Figure S25A). To explore the targeting effect of peptide, the myocardial ischemic mice were also injected intravenously with BDP-NO2@Lip without peptide functionalization and subjected to imaging (Figure 4A). Although the heart displayed gradually increased fluorescence, the interferences from normal tissues such as lung, liver were serious, leading to a low fluorescence signal and signal to noise ratio at all time points (Figure S25A). Overall, the contribution of AT1 to targeting and the fluorescence sensitivity of BDP-NO2 to hypoxia resulted in successful in vivo myocardial ischemia imaging by Pep/BDP-NO2@Lip. For a more detailed examination, the heart and other major organs of the nanoprobe-treated mice were harvested for ex vivo imaging after 12 h post ligation. Ex vivo imaging (Figure 4B) and semiquantitative analysis (Figure S25B) of Pep/BDPNO2@Lip distribution clearly showed that specific activation of BDP-NO2 mainly occurred at the ischemic heart tissue over other organs including liver, spleen, lung, kidneys, large intestine and small intestine. The high contrast for heart imaging was attributed to two main reasons. First, a targeted delivery of the nanoprobe to heart due to the presence of targeted peptide at their surface and AT1 receptors overexpressed on cardiac cells. Second, the fluorescence of the loaded molecular BDP-NO2 was very weak, the off-on fluorescence response occurred only after it being released and reduced under suitable conditions. On the contrary, the BDPNO2@Lip treated excised tissues showed that fluorescence signal was observed in heart, but the high fluorescence background was also observed in liver, spleen, lung and kidneys, indicating poor ability to target. In addition, the fluorescence was also detected in faces in intestines (Figure S26), indicating that nanoprobe could be excreted out from the body, which reduced the potential side effects in body. In addition, hematoxylin and eosin (H&E) staining of the different treatment groups was performed to investigate the pathological change of myocardial tissue (Figure 4C). In control (without any operations) and sham operation groups, the cardiac muscle fibers were relatively uniform, with little inflammatory infiltration, edema or cardiac necrosis, which


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suggested that nanoprobe itself showed no damage to the heart of mice. However, the mice in the myocardial ischemia model group displayed that myocardial fibers were in disordered and

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Figure 4. (A) Time-dependent in vivo fluorescence imaging of myocardial ischemic mice after intravenous injection of Pep/BDP-NO2@Lip or BDP-NO2@Lip, the time shown in the figure indicates the time after arterial ligation. (B) Representative fluorescence images of harvested organs from mice upon different treatments at 12 h after surgery. (C) Representative microscopic images of H&E staining of myocardial tissue sections from different treatment groups at 12 h after surgery. Scale bars: 100 μm.

wavy arrangement and showed fracture, sarcoplasm swelling, increased eosinophils, and obvious inflammation cell infiltration even contraction band necrosis. The result implied that the model was successful and Pep/BDP-NO2@Lip could indeed indicate hypoxia in myocardial ischemia process specifically. The serum markers of myocardial injury are one of the important indicators for clinical diagnosis of myocardial ischemic injury.46 Thus, CK, CK-MB, cTnI, and Mb as clinical examination biomarkers were used to assess the myocardial injury after 12 h arterial ligation in mice of each group (Figure S27). Compared with the control group and the sham group, the content of the four serum markers in the two model group increased significantly, indicative of a successful establishment of myocardial ischemia model. Taken together, it demonstrated that the fluorescence signal of heart was associated with myocardial damage.

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The potential toxicity or side effects are always a great concern for nanoprobes applied in vivo. At 7 days after the treatment, the age-matched healthy mice without treatment (control) and the Pep/BDP-NO2@Lip treated healthy mice were sacrificed, and the major organs including heart, liver, spleen, lung, and kidney were collected for H&E staining to assess side effects. No abnormalities were observed in organ tissues for nanoprobe-treated group compared with the control (Figure S28), confirming the negligible toxic side effects of Pep/BDP-NO2@Lip for in vivo applications. CONCLUSIONS In summary, we have successfully developed Pep/BDPNO2@Lip, a myocardium-targeted, fluorescent off-on BDPNO2 encapsulated NIR nanoprobe that detects hypoxia degree in myocardial cells and tissues. The targeting ability is achieved by taking advantage of the overexpression of AT1 receptor in the ischemic heart. Our approach is based on the ability of NTR to catalyze the reduction of BDP-NO2 with excellent selectivity. After myocardial hypoxia or ischemia, the nanoprobe conjugated with a ligand specific for the AT1 receptor do specifically target the heart, where they can release the BDPNO2. The nitro group of BDP-NO2 is reduced by NTR to generate amino-compound, leading to the inhibition of an electro-withdrawing group induced electron-transfer process and following a fluorescence enhancement. At the cellular level, confocal fluorescence imaging of hypoxic H9c2 cells demonstrates the NTR detection ability of the nanoprobe. Furthermore, the nanoprobe has been successfully used for realtime imaging of the degrees of hypoxia in mouse model of myocardial ischemia, further highlighting the potential diagnostic application of it. To our knowledge, this work is the first to utilize NTR as a sensitive biomarker to detect myocardial hypoxia by fluorescence with a low detection limit of 7.08 ng/mL. Our newly NIR fluorescent off-on system exhibits high selectivity and sensitivity, good biocompatibility and simplicity properties, which may provide new opportunities for noninvasive in vivo myocardial hypoxia detection, as well as great assistance in ischemic heart disease diagnosis. Moreover, we will develop molecular probes in the second near-infrared window (NIR-II) in later work, which is expected to overcome the challenge of imaging depth.

74 ASSOCIATED CONTENT 75 Supporting Information. 76 Supplemental experiments, tables, figures and references. This 77 material is available free of charge via the Internet at 78 http://pubs.acs.org. 79 AUTHOR INFORMATION 80 Corresponding Author 81 E-mail: [email protected] 82 E-mail: [email protected] 83 84 ORCID 85 Bo-Yang Yu: 0000-0002-1401-9250

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Jiangwei Tian: 0000-0003-1018-4694

Author Contributions ‡Y.

F. and M. L. contributed equally to this work.

Notes The authors declare no competing financial interest.

8 ACKNOWLEDGMENT 9 This research was supported by National Natural Science 10 Foundation of China (21775166), Natural Science Foundation 11 for Distinguished Young Scholars of Jiangsu Province 12 (BK20180026), “Double First-Class” University Project 13 (CPU2018GF06, CPU2018GY32), and Six Talent Peaks 14 Project in Jiangsu Province. The authors acknowledge Dr. 15 Robert Paugh for help in English writing. 16 17 REFERENCES 18 (1) Noda, H.; Iso, H.; Saito, I.; Konishi, M.; Inoue, M.; Tsugane, 19 S. The Impact of the Metabolic Syndrome and Its Components on 20 the Incidence of Ischemic Heart Disease and Stroke: the Japan 21 Public Health Center-Based Study. Hypertens Res. 2009, 32, 28922 298. 23 (2) Knudtson, M. L.; Wyse, D. G.; Galbraith, P. D.; Brant, R.; 24 Hildebrand, K.; Paterson, D.; Richardson, D.; Burkart, C.; Burgess, 25 E. Chelation Therapy for Ischemic Heart Disease: a Randomized 26 Controlled Trial. JAMA. 2002, 287, 481-486. 27 (3) Eltzschig, H. K.; Bonney, S. K.; Eckle, T. Attenuating 28 Myocardial Ischemia by Targeting A2B Adenosine Receptors. 29 Trends Mol. Med. 2013, 19, 345-354. 30 (4) Lee, S. H.; Wolf, P. L.; Escudero, R.; Deutsch, R.; Jamieson, 31 S. W.; Thistlethwaite, P. A. Early Expression of Angiogenesis 32 Factors in Acute Myocardial Ischemia and Infarction. N. Engl. J. 33 Med. 2000, 342, 626-633. 34 (5) Verdouw, P. D.; van. Den. Doel, M. A.; de. Zeeuw, S.; 35 Duncker, D. J. Animal Models in the Study of Myocardial Ischaemia 36 and Ischaemic Syndromes. Cardiovasc. Res. 1998, 39, 121-135. 37 (6) Azzouzi, H. E.; Leptidis, S.; Doevendans, P. A.; De. Windt, L. 38 J. HypoxamiRs: Regulators of Cardiac Hypoxia and Energy 39 Metabolism. Trends Endocrinol. Metab. 2015, 26, 502-508. 40 (7) Jopling, C.; Suñé, G.; Faucherre, A.; Fabregat, C.; Izpisua 41 Belmonte, J. C. Hypoxia Induces Myocardial Regeneration in 42 Zebrafish. Circulation 2012, 126, 3017-3027. 43 (8) Brown, J. M.; Wilson, W. R. Exploiting Tumour Hypoxia in 44 Cancer Treatment. Nat. Rev. Cancer 2004, 4, 437-447. 45 (9) Liu, Z. R.; Tang, Y.; Xu, A.; Lin, W. A New Fluorescent Probe 46 with a Large Turn-On Signal for Imaging Nitroreductase in Tumor 47 Cells and Tissues by Two-Photon Microscopy. Biosens. Bioelectron. 48 2017, 89, 853-858. 49 (10) Ao, X.; Bright, S. A.; Taylor, N. C.; Elmes, R. B. P. 250 Nitroimidazole Based Fluorescent Probes for Nitroreductase; 51 Monitoring Reductive Stress in Cellulo. Org. Biomol. Chem. 2017, 52 15, 6104-6108. 53 (11) Parkinson, G. N.; Skelly, J. V.; Neidle, S. Crystal Structure 54 of FMN-Dependent Nitroreductase from Escherichia coli B: A 55 Prodrug-Activating Enzyme. J. Med. Chem. 2000, 43, 3624-3631. 56 (12) Okuda, K.; Okabe, Y.; Kadonosono, T.; Ueno, T.; Youssif, 57 B. G.; Kizaka-Kondoh, S.; Nagasawa, H. 2-Nitroimidazole58 Tricarbocyanine Conjugate as a Near-Infrared Fluorescent Probe 59 for in Vivo Imaging of Tumor Hypoxia. Bioconjug. Chem. 2012, 23, 60 324-329.

61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124

Page 8 of 10

(13) Handley, M. G.; Medina, R. A.; Nagel, E.; Blower, P.J.; Southworth, R. PET Imaging of Cardiac Hypoxia: Opportunities and Challenges. J. Mol. Cell. Cardiol. 2011, 51, 640-650. (14) Lapi, S. E.; Voller, T. F.; Welch, M. J. PET Imaging of Hypoxia. PET Clin. 2009, 4, 39-47. (15) Strauss, H. W.; Nunn, A.; Linder, K. Nitroimidazoles for Imaging Hypoxic Myocardium. J. Nucl. Cardiol. 1995, 2, 437-445. (16) Sinusas, A. J. The Potential for Myocardial Imaging with Hypoxia Markers. Semin. Nucl. Med. 1999, 29, 330-338. (17) Nunn, A.; Linder, K.; Strauss, H. W. Nitroimidazoles and Imaging Hypoxia. Eur. J. Nuel. Med. 1995, 22, 265-280. (18) Tian, Z.; Li, A. D. Photoswitching-Enabled Novel Optical Imaging: Innovative Solutions for Real-World Challenges in Fluorescence Detections. Acc. Chem. Res. 2013, 46, 269-279. (19) Zhang, G.; Palmer, G. M.; Dewhirst, M. W.; Fraser, C. L. A Dual-Emissive-Materials Design Concept Enables Tumour Hypoxia Imaging. Nat. Mater. 2009, 8, 747-751. (20) Chen, H. J.; Chew, C. Y.; Chang, E. H.; Tu, Y. W.; Wei, L. Y.; Wu, B. H.; Chen, C. H.; Yang, Y. T.; Huang, S. C.; Chen, J. K.; Chen, I. C.; Tan, K. T. S-Cis Diene Conformation: A New Bathochromic Shift Strategy for Near-Infrared Fluorescence Switchable Dye and the Imaging Applications. J. Am. Chem. Soc. 2018, 140, 5224–5234. (21) Kowada, T.; Maeda, H.; Kikuchi, K. BODIPY-Based Probes for the Fluorescence Imaging of Biomolecules in Living Cells. Chem. Soc. Rev. 2015, 44, 4953-4972. (22) Kamkaew, A.; Lim, S. H.; Lee, H. B.; Kiew, L. V.; Chung, L. Y.; Burgess, K. BODIPY Dyes in Photodynamic Therapy. Chem. Soc. Rev. 2013, 42, 77-88. (23) Niu, L.Y.; Guan, Y. S.; Chen, Y. Z.; Wu, L. Z.; Tung, C. H.; Yang, Q. Z. BODIPY-Based Ratiometric Fluorescent Sensor for Highly Selective Detection of Glutathione over Cysteine and Homocysteine. J. Am. Chem. Soc. 2012, 134, 18928-18931. (24) Rurack, K.; Kollmannsberger, M.; Daub, J. Molekulares Schalten im nahen Infrarot (NIR) mit einem funktionalisierten Bordipyrromethen-Farbstoff. Angew. Chem. 2001, 113, 396-399. (25) Rurack, K.; Kollmannsberger, M.; Daub, J. A Highly Efficient Sensor Molecule Emitting in The Near Infrared (NIR): 3, 5-Distyryl-8-(p-Dimethylaminophenyl)difluoroboradiaza-sindacene. New J. Chem. 2001, 25, 289-292. (26) Allen, T. M.; Cullis, P. R. Liposomal Drug Delivery Systems: From Concept to Clinical Applications. Adv. Drug Deliv. Rev. 2013, 65, 36-48. (27) Molavi, B.; Chen, J.; Mehta, J. L. Cardioprotective Effects of Rosiglitazone are Associated with Selective Overexpression of Type 2 Angiotensin Receptors and Inhibition of p42/44 MAPK. Am. J. Physiol. Heart Circ. Physiol. 2006, 291, H687-693. (28) Yang, Z.; Bove, C. M.; French, B. A.; Epstein, F. H.; Berr, S. S.; DiMaria, J. M.; Gibson, J. J.; Carey, R. M.; Kramer, C. M. Angiotensin II Type 2 Receptor Overexpression Preserves Left Ventricular Function after Myocardial Infarction. Circulation 2002, 106, 106-111. (29) Greene, L. J.; Spadaro, A. C.; Martins, A. R.; Perussi, De. Jesus, W. D.; Camargo, A. C. Brain Endo-Oligopeptidase B: a PostProline Cleaving Enzyme that Inactivates Angiotensin I and II. Hypertension 1982, 4, 178-184. (30) Weis, S. M. Vascular Permeability in Cardiovascular Disease and Cancer. Curr. Opin. Hematol. 2008, 15, 243-249. (31) Muthu, M. S.; Kulkarni, S. A.; Xiong, J.; Feng, S. S. Vitamin E TPGS Coated Liposomes Enhanced Cellular Uptake and Cytotoxicity of Docetaxel in Brain Cancer Cells. Int. J. Pharm. 2011, 421, 332-340. (32) Tani, M.; Neely, J. R. Role of Intracellular Na+ in Ca2+ Overload and Depressed Recovery of Ventricular Function of


Page 9 of 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Reperfused Ischemic Rat Hearts. Possible Involvement of H+-Na+ and Na+-Ca2+ Exchange. Circ. Res. 1989, 65, 1045−1056. (33) Jacobus, W. E.; Taylor, G. J. IV; Hollis, D. P.; Nunnally, R. L. Phosphorus Nuclear Magnetic Resonance of Perfused Working Rat Hearts. Nature 1977, 265, 756-758. (34) Xu, J.; Sun, S.; Li, Q.; Yue, Y.; Li, Y.; Shao, S. A Rapid Response “Turn-On” Fluorescent Probe for Nitroreductase Detection and Its Application in Hypoxic Tumor Cell Imaging. Analyst 2015, 140, 574-581. (35) Li, Z.; He, X.; Wang, Z.; Yang, R.; Shi, W.; Ma, H. In Vivo Imaging and Detection of Nitroreductase in Zebrafish by a New Near-Infrared Fluorescence off-on Probe. Biosens. Bioelectron. 2015, 63, 112-116. (36) Yang, D.; Tian, H. Y.; Zang, T. N.; Li, M.; Zhou, Y.; Zhang, J. F. Hypoxia Imaging in Cells and Tumor Tissues Using a Highly Selective Fluorescent Nitroreductase Probe. Sci. Rep. 2017, 7, 9174. (37) Li, Z.; Li, X.; Gao, X.; Zhang, Y.; Shi, W.; Ma, H. Nitroreductase Detection and Hypoxic Tumor Cell Imaging by a Designed Sensitive and Selective Fluorescent Probe, 7-[(5Nitrofuran-2-yl)methoxy]-3H-phenoxazin-3-one. Anal. Chem. 2013, 85, 3926-3932. (38) Maeda, H.; Nakamura, H.; Fang, J. The EPR Effect for Macromolecular Drug Delivery to Solid Tumors: Improvement of Tumor Uptake, Lowering of Systemic Toxicity, and Distinct Tumor Imaging in Vivo. Adv. Drug Deliv. Rev. 2013, 65, 71-79. (39) Regitz, V.; Paulson, D. J.; Hodach, R. J.; Little, S. E.; Schaper, W.; Shug, A. L. Mitochondrial Damage during Myocardial Ischemia. Basic Res. Cardiol. 1984, 79, 207-217.

29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

(40) Borutaite, V.; Jekabsone, A.; Morkuniene, R.; Brown, G. C. Inhibition of Mitochondrial Permeability Transition Prevents Mitochondrial Dysfunction, Cytochrome c Release and Apoptosis Induced by Heart Ischemia. J. Mol. Cell. Cardiol. 2003, 35, 357-366. (41) Feng, P.; Zhang, H.; Deng, Q.; Liu, W.; Yang, L.; Li, G.; Chen, G.; Du, L.; Ke, B.; Li, M. Real-Time Bioluminescence Imaging of Nitroreductase in Mouse Model. Anal. Chem. 2016, 88, 5610-5614. (42) Vengellur, A.; Woods, B. G.; Ryan, H. E.; Johnson, R. S.; LaPres, J. J. Gene Expression Profiling of the Hypoxia Signaling Pathway in Hypoxia-Inducible Factor 1α Null Mouse Embryonic Fibroblasts. Gene Expr. 2003, 11, 181-197. (43) Fang, Z.; Luo, W.; Luo, Y. Protective Effect of α-Mangostin against CoCl2-Induced Apoptosis by Suppressing Oxidative Stress in H9c2 Rat Cardiomyoblasts. Mol. Med. Rep. 2018, 17, 6697-6704. (44) Wang, Z.; Liao, S. G.; He, Y.; Li, J.; Zhong, R. F.; He, X.; Liu, Y.; Xiao, T. T.; Lan, Y. Y.; Long, Q. D.; Wang, Y. L. Protective Effects of Fractions from Pseudostellaria Heterophylla against Cobalt Chloride-Induced Hypoxic Injury in H9c2 Cell. J. Ethnopharmacol. 2013, 147, 540-545. (45) Reichert, K.; Colantuono, B.; Mccormack, I.; Rodrigues, F.; Pavlov, V.; Abid, M. R. Murine Left Anterior Descending (LAD) Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction. J. Vis. Exp. 2017, 122, 1-6. (46) Bodor, G. S. Biochemical Markers of Myocardial Damage. Clin. Chem. Lab. Med. 2016, 27, 95-111.



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Targeted Myocardial Hypoxia Imaging Using a Nitroreductase

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