A Highly Specific and Sensitive Radioiodinated Agent for in vivo

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A Highly Specific and Sensitive Radioiodinated Agent for in vivo Imaging of Superoxide Through Superoxide-initiated Retention Lumei Huang, Zijing Li, Deliang Zhang, Hua Li, Changrong Shi, Pu Zhang, Xinhui Su, and Xianzhong Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03642 • Publication Date (Web): 11 Oct 2018 Downloaded from http://pubs.acs.org on October 12, 2018

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Analytical Chemistry

A Highly Specific and Sensitive Radioiodinated Agent for in vivo Imaging of Superoxide Through Superoxide-initiated Retention Lumei Huang1, Zijing Li1,*, Deliang Zhang1, Hua Li1, Changrong Shi1, Pu Zhang1, Xinhui Su2, Xianzhong Zhang1,*

1State

Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & Center for

Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen 361102, Fujian, China. 2Zhongshan

Hospital Xiamen University, Xiamen 361004, Fujian, China.

Running title: in vivo imaging of superoxide First author: Lumei Huang, Ph. D. candidate, Center for Molecular Imaging and Translational Medicine, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiang’An South Rd. No. 422116, Xiang’An district, Xiamen 361102, China. Email: [email protected], phone: +86-592-2880641. * Corresponding authors: Xianzhong Zhang Ph. D. and Zijing Li, Ph. D., Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiang’An South Rd. No. 422-116, Xiang’An District, Xiamen 361102, China. Xianzhong Zhang, email: [email protected], phone: +86-592-2880645. 1

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Analytical Chemistry 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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Zijing Li, email: [email protected], phone: +86-592-2880643.

Abstract Superoxide (O2•−) is a specific molecular target for xenobiotics, cytokines and bacteria during inflammatory diseases. The aim of this study is to develop a single-photon emission computed tomography (SPECT) imaging agent and to quantify the distribution of O2•− in vivo.

125/131I-PISO

was obtained in good radiochemical yield (60-70%) and high

radiochemical purity (> 98%) after HPLC purification. 125/131I-PISO (log P = 2.46) could be oxidized by O2•− selectively and sensitively, converted to a hydrophilic compound 125/131I-PISA

(log P = -1.62) with negative charge simultaneously and conglutinated with

biomolecules by electrostatic interactions. The specific accumulation of

131I-PISA

in the

O2•− rich region were verified in cell efflux assay and SPECT/CT imaging in situ O2•− enrichment model mice. SPECT/CT imaging showed higher accumulation of 125I-PISO in the inflamed ankles compared to the control. Radioiodinated PISO is a potential SPECT agent to image O2•− distribution in vivo through specific and sensitive O2•− triggered retention.

Keywords: radioiodinated tracer, superoxide, SPECT/CT, polarity inversion, in vivo imaging.

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Introduction Superoxide (O2•−), a major member of the reactive oxygen species (ROS) family, is the one-electron reduced form of oxygen that is the indispensable terminal receptor of all electrons in the living subjects 1. As an electronating anionic inflammatory mediator upregulated under certain stimulation to drive necroptosis and apoptosis, lipophobic O2•− has very limited perfusion through the cell membrane 2 and thus accumulates in the focus area to destroy intruders like bacteria, viruses, the body's own damaged cells and macromolecules 3. Therefore, O2•− has been recognized as a specific molecular target that regulates various signaling cascades, for instance, hypoxic tumors 4, chronic inflammation 5-7,

ischemia 8, tissue injury 9, fibrosis 10, remodeling 11 and organ dysfunction 12. Even in

mild chronic inflammation such as varicosity, the mean O2•− radical concentration in the tissue under ROS stress (69.5 ± 11.9 nmol/mL) was also significantly higher than that in normal (33.8 ± 10.5 nmol/mL, P < 0.05) 13. O2•− was also the primary acting molecule and direct functional performer of chemotherapy, radiotherapy, photodynamic therapy, during which much higher exogenous O2•− concentration needed for effective cell killing 14, and overdosing is prevailing to ensure the effectiveness 15. Nevertheless, the exact O2•− distribution and intensity during these diseases and therapies are difficult to measure in vivo for its high reactivity and short half-life

16,17.

Hence, a

feasible method to selectively and quantitatively image either endogenous or exogenous

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O2•− in intact animals or patients in real time will be very helpful to understand the links between ROS and diseases. Current ROS imaging techniques include fluorescence imaging 18,19, photoacoustic (PA) 2022,

electron paramagnetic resonance imaging

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and nuclear imaging. Nuclear imaging

modalities such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) have been most widely applied to monitor very low amount of targets in completely living organisms at acceptable spatial resolutions. The spatiotemporal changes of ROS generation after ischemia/reperfusion was once investigated with the radical trapping radiotracer [3H]hydromethidine 24. Chu et al. reported a 18F labeled dihydroethidium analog as a reductive PET tracer to imaging ROS 25. 18F-5fluoro-l-aminosuberic acid was developed with the goal of assessing its potential as a diagnostic tracer of oxidative stress via xC-activity 26,27. Carroll et al. have developed 11CVitC that exhibits ROS-dependent cellular accumulation 28. Salma Al-Karmi et al. prepared a 18F labeled hydrocyanine dye following the reaction with hydroxyl radicals or superoxide, represents an example of multimodal PET and “turn-on” near IR type ROS probe 29. More recently, Hou et al. synthesized a neuroinflammation murine model

30.

18F-ROStrace

to image superoxide levels in a

Up today, most of the fluorescent and PET probes

were developed basing on a redox strategy to detect ROS, and innovative specific probe with higher sensitivity to O2•− is still required for more precise mapping of oxidative stress.

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Contemporarily, optical probes progressed fast and three novel fluorescent probes via a non-redox strategy for ultra-sensitive and ultra-selective detection of O2•− in intact live zebrafish embryos was reported 31. Inspired by this mechanism, an activatable fluorophore (5-iodo-3-oxo-3H-spiro[isobenzofuran-1,9’-xanthene]-3’,6’-diylbis(trifluoromethane sulfonate) (named PISO) and its radioiodinated probe (125/131I-PISO, Figure 1) were synthesized in this study. The selectivity and sensitivity of PISO and

125/131I-PISO

for

superoxide were verified in vitro. The hypothesis of “polarity-inversing then trapped” nonredox strategy for specific detection of O2•− was assessed by cell efflux experiments and SPECT imaging with model mice. SPECT imaging revealed the biodistribution of O2•− in living subjects semi-quantitatively. Our probe thus for the first time enable in vivo evaluation of superoxide by whole-body SPECT/CT imaging simultaneously.

Experimental Section General Materials All reagents used in this study were purchased from J&K Scientific Ltd. (China) and used without further purification. Reactions were monitored with thin layer chromatography on Silica gel 60 F254 aluminum sheets (Merck, Germany), with compounds visualized with a 254 nm UV lamp. Column chromatography purifications were performed with 54 - 74 μm silica gel (Qingdao Haiyang Chemical Co. Ltd., China).

131I-NaI

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aqueous solution were


obtained from Zhongshan Hospital affiliated of Xiamen University.

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125I-NaI

were

purchased from China Isotope & Radiation Corporation (Beijing, China). Instruments 1H

NMR and 13C NMR characterization of our synthesized compounds dissolved in CDCl3

or DMSO-d6 were measured on a 600 MHz Spectrometer (Avance II, Bruker, Germany), with tetramethylsilane as the internal standard. High-resolution mass spectrometry was acquired on a Q Exactive LC-MS system (Waters, America). Fluorescence spectrometry was recorded by a Perkin Elmer LS55 fluorescence spectrophotometer (Perkin Elmer, America). HPLC analysis and purification were performed on a Thermo Scientific Dionex Ultimate 3000 system equipped with a SPD-20A UV detector as well as a Flow Count 3200 NaI/PMT γ-radiation scintillation detector (Bioscan, USA). Mobile phase A, ultrapure water. Phase B, HPLC grade acetonitrile (Amethyst Chemicals, China). Thermo Scientific Hypersil Gold columns (250 × 4.6 mm, 250 × 10 mm) were employed in isocratic elution with 75% B at a flow rate of 1.5 mL/min for analysis. MicroSPECT/CT imaging was acquired with a nanoScan SC (Mediso Medical Imaging System, Hungary). Fluorescent imaging was performed on a Carestream Fx Pro/FX imaging system (Carestream Health Inc.). Ex vivo fluorescence staining was performed with an inverted microscope Ti-U (Nikon, Japan). Radioactivities were measured on 2480 Wizard2 Automatic Gamma Counter (Perkin Elmer, USA). The centrifuge we used in log P

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determination was Eppendorf Centrifuge 5424 R (Eppendorf, Germany). Animal Models The experimental procedures and the animal use and care protocols were approved by the Institutional Animal Care and Use Committee of Xiamen University. All experimental protocols were carried out in accordance with the relevant guidelines. Hind paw inflammation model. ICR mice (5 weeks, male, 20 g) obtained from the Laboratory Animal Center of Xiamen University were injected subcutaneously with 50 μL of complete Freund’s adjuvant (CFA Sigma-Aldrich) to induce inflammation reaction in one hind paw and were monitored for 7 days 32. The inflamed areas were also visualized by magnetic resonance imaging. All mice were maintained on rodent chow, and housed in a standard 12 h light-dark cycle for the duration of the study. Liver fibrosis model. C57BL/6 mice (6 weeks, female, 20 g) were administered 100 μL of a 40% solution of CCl4 (Sigma, St. Louis, MO) in olive oil by intraperitoneal injection, two times a week for 8 weeks to induce fibrosis. Controls received only pure olive oil 33. Animals were imaged one week after the last injection to avoid acute effects of CCl4. In situ O2•− enrichment model. ICR mice (5 weeks, male, 20 g) obtained from the Laboratory Animal Center of Xiamen University were intraperitoneal (i.p.) injected with 100 μL saline (control group) or X/XO solution (experimental group) to induce O2•−

34,

while the blocking study followed by injection of Tiron (a specific O2•− scavenger, 0.4 μg/g 7



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body weight). In situ model mice with graded O2•−conc. were induced by X/XO of different concentrations: group 1, X/XO = 3 μM/30 mU; group 2, X/XO = 9 μM/90 mU; mice of group 3 were given twice i.p. injections of graded dosages of X/XO in separated areas, left side, X/XO = 3 μM/30 mU, right side, X/XO = 9 μM/90 mU, n = 3 for each group. LPS treated model was performed with ICR mice (5 weeks, male, 20 g) i.p. injected with either low (0.6 μg/g of body weight) or high concentration LPS (in saline, 1.2 μg/g body weight) and saline (control group) for 60 days

35.

Tiron (0.4 μg/g body weight) was

administered to high dosage LPS-treated mice 6 h before measurement for blocking study 36.

MicroSPECT/CT Imaging Protocol MicroSPECT/CT imaging was acquired with a nanoScan SC (Mediso Medical Imaging System, Hungary) equipped with pinhole collimator following standard animal scan procedure (CT acquiring parameters: energy peak of 50 kV, 670 μA, 480 projections, medium zoom; SPECT acquiring parameters: energy peak of 364 keV for imaging, 28 keV for

125I-PISO

131I-PISO

imaging, window width of 20%, matrix of 256 × 256,

medium zoom, 30 s frame).

Chemistry and Radiochemistry 5/6-bromo-3',6'-dihydroxy-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one (1:1) (1) 5-bromoisobenzofuran-1,3-dione (0.454 g, 2.0 mmol) and resorcinol (0.44 g, 4.0 mmol)

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were dissolved in 5 mL methanesulfonic acid. The mixed solution then heated to 120 °C for 2 h before cooling to room temperature. Then the reaction mixture was poured into water tardily and concentrated under vacuum to precipitate solid product. The precipitation was washed with water 3 times and dried to obtain 0.738 g product at a yield of 90%. 1H NMR (600 MHz, DMSO-d6): δ 6.52 (d, 2H, J = 5.31 Hz) δ: 6.672 (s, 2H), 7.251 (d, 2H, J = 2.4 Hz), 7.91(d, 1H, J = 6.0 Hz), 7.95 (d, 1H, J = 1.8 Hz), 8.15(s, 1H), 10.15(s, 2H). 13CNMR (150 MHz, DMSO-d6) δ 102.7, 109.4, 113.2, 123.6, 125.8, 127.5, 128.9, 129.6, 129.7, 133.9, 138.8, 152.3, 160.1, 167.6 HRMS (FTMS ESI+): m/z calculated for C20H12BrO5+: 410.9868, found 410.9871 [M]+. 5-bromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl bis(trifluoromethanesulfonate) (2) 1 (0.205 g, 0.5 mmol) and 2 mL triethylamine were dissolved in 10 mL anhydrous dichloromethane in a 50 mL double neck bottle. A solution of Trifluoromethanesulfonic anhydride in anhydrous dichloromethane (0.423 g, 1 mmol) was added dropwise at -78 °C under nitrogen. Then the reaction mixture was stirred for 20 min at -78 °C and 20 min at 25 °C. The mixture was dilute with acetic ether, then washed with 1 M HCl, water and saturated NaCl, respectively for three times. Organic phase was dried with anhydrous magnesium sulfate. Evaporated under vacuum, the resulting residue was purified by column chromatography on silica gel ethyl with acetate/petroleum ether 1: 8 to yield 2

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(0.532 g, 79%). 1H NMR (600 MHz, CDCl3), δ: 7.00 (d, 2H, J = 6.00 Hz), 7.08 (d, 1H, J = 6.02 Hz), 7.10 (d, 2H, J = 6.30 Hz), 7.33 (s, 2H), 7.88 (d, 1H, J = 12.00 Hz), 8.23 (s, 1H). 13C-NMR (150 MHz, CDCl3), δ: 80.2, 110.8, 117.8, 118.6, 119.7, 125.0, 125.2, 127.5, 128.6, 129.8, 138.9, 150.3, 150.7, 151.2, 166.8. HRMS (FTMS ESI+): m/z calculated for C22H9BrF6NaO9S2+: 696.8673, found 696.8680 [M]+. 3-oxo-5-(tributylstannyl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl bis(trifluoromethanesulfonate) (3) 2 (0.15 g, 0.22 mmol) was added into a round bottom bottle and dissolved with 5.0 mL toluene. Hexabutylditin (0.3 g, 0.517 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.002 g) were added into the solution. The reaction mixture was stirred at 120 °C under refluxing for 1.5 h. The reaction mixture was filtered, concentrated to 2 mL under vacuum and directly loaded onto a silica gel column. The column was eluted with ethyl acetate/petroleum ether (1:2), and the collected component was evaporated under vacuum to give a pale-yellow solid product (0.15 g, 17%). 1H NMR (600 MHz, CDCl3), δ: 0.921 (m, 9H), 1.311(m, 6H), 1.388 (m, 6H), 1.591 (m, 6H), 6.99 (d, 2H, J = 6.00 Hz), 7.06 (d, 2H, J = 3.01), 7.14 (s, 1H), 7.31 (s, 2H), 7.82 (d, 1H, J = 6.00 Hz), 8.23 (s, 1H). 13C-NMR (150 MHz, CDCl3), δ:13.5, 16.4, 26.6, 29.2, 79.9, 110.6, 117.6, 119.7, 122.7, 123.9, 124.4, 124.7, 130.1, 133.2, 143.4, 150.2, 151.3, 151.8, 169.1. HRMS (FTMS ESI+): m/z calculated for C34H37F6O9S2Sn+: 887.0805, found 887.0812 [M]+.

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5-iodo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl bis(trifluoromethanesulfonate) (4) 3 (0.05 g, 0.056 mmol) and iodine (0.025 g, 0.1 mmol) were dissolved in 1.0 mL anhydrous THF and 1 mL water in a 10 mL serum vial. After stirring at room temperature for 1 h, the mixture was extracted with ethyl acetate (2 × 5 mL). Organic layers were combined and dried with anhydrous sodium sulfate. Evaporated under vacuum, the resulting residue was purified by column chromatography on silica gel (ethyl acetate/petroleum ether = 1:10) to yield 5 (0.038 g, 76%). 1H NMR (600 MHz, CDCl3), δ: 6.96 (d, 1H, J = 7.81 Hz), 7.00 (d, 2H, J = 6.00 Hz), 7.07 (d, 2H, J = 2.40 Hz), 7.32 (d, 2H, J = 2.40 Hz), 8.07 (d, 2H, J = 12.00 Hz), 8.44 (s, 1H). 13C-NMR (150 MHz, CDCl3), δ: 80.2, 110.8, 117.7, 118.7, 119.7, 125.4, 127.6, 129.9, 133.8, 134.7, 144.6, 150.3, 151.2, 151.4, 166.7. HRMS (FTMS ESI+): m/z calculated for C22H10F6IO9S2+: 722.8715, found 722.9020 [M]+. Radioiodination Precursor (3, 0.05 mg) was dissolved with anhydrous acetonitrile (100 μL) in a glass vial. Chloramine-T (100 μL, 1.0 mg/mL), 131I-NaI (20 μL, 2.0 mCi) or 125I-NaI (15 μL, 2.0 mCi) and HCl (100 μL, 1.0 mol/L) were added into the vial successively. The mixture was kept at room temperature for 15 min before quenching with a saturated sodium metabisulfite solution. The radioiodination product was analyzed by radio-HPLC and co-injected with the non-radioactive reference compound PISO. After HPLC purification (75% acetonitrile

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and 25% water, 1.5 mL/min), the collected component was dried with N2 flow and dissolved in saline mixed with 5% ethanol (v/v) for further use. Selectivity and Reactivity of PISO for O2·PISO (15 μM) dissolved in phosphate buffer at pH 7.4 (0.1 mol/L, 5% ethanol) was incubated respectively with KO2 (15 μM), several typical reactive oxygen species (50 mM H2O2, .OH, NO, 1O2, HOCl), reductive amino acids (50 mM Gly, Pro and His), natural reductants (50 mM GSH and vitamin C), and metal ions (50 mM Cl-, F-, Mn2+, Fe3+ and Fe2+) for 1 h at 25 °C. Fluorescence intensities were measured by a LS55 spectrophotometer (Perkin Elmer, USA). The procedure of reactivity study was described in the supplemental information. Specific Activities Various concentrations of PISO solution (0.01, 0.05, 0.1, 0.25, 0.5, 1, 5 mg/mL) were prepared and analyzed by an analytical HPLC (isocratic elution, flow rate: 1.5 mL/min, A: 25% pure water B: 75% ACN). The UV absorption peak areas at different concentrations of PISO were measured and the relationship between the concentration of the substance and the area of the absorption peak was obtained by linear analysis. Partition Coefficient Partition coefficients (log P) of

131I-PISO

were measured in 1-octanol and phosphate

buffered saline (PBS, pH = 7.4), both pre-equilibrated with each other for 14 days. The

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procedure was as follows: 0.37 MBq 131I-PISO in water (100 μL) after HPLC purification was added in a plastic centrifuge tube to which 1.9 mL of PBS and 2 mL 1-octanol was added followed. The tube was vortexed for 5 min followed by 5 min of centrifugation at 12,000 rpm. Aliquots of 100 μL from each phase were measured in an automatic gamma counter. Partition coefficient was expressed as log P was calculated as the formula: P = Counts in 1-octanol-background/Counts in PBS-background. The experiment was performed in triplicate. The log P of 131I-PISA was measured following the above method. Stability and Metabolic Analysis Purified 131I-PISO (~3.7 MBq) was incubated in 500 μL saline at room temperature. The radiochemical purity (RCP) was assayed by HPLC at 30 min and 1 h. Stability in serum was determined by incubating ~3.7 MBq purified

131I-PISO

compound in the solution of

500 μL bovine serum at 37 °C for 30 min and 1 h. the mixture was precipitated by addition of 100 μL acetonitrile and removed by centrifugation at 12, 000 rpm for 5 min at room temperature. After filtered by the 0.22 μm Millipore filter, the radiochemical purity was analyzed by HPLC. Metabolic stability of 131I-PISO were performed according to the published procedure. ICR mice were injected with purified

131I-PISO

(37 MBq) through the tail vein. Mice were

sacrificed at 30 min and 1 h, and then plasma and urine were collected respectively. Plasma samples were added with 100 μL acetonitrile immediately and centrifuged at 12,000 rpm

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for 5 min. Urine sample was directly diluted with 1.0 mL of phosphate-buffered saline (PBS, 0.025 M, pH 7.4). All samples were passed through a 0.22 µm Millipore filter and 20 µL of the supernatants were analyzed by HPLC respectively.

Cell Uptakes and Efflux of 131I-PISO RAW264.7 cells were obtained from American Type Culture Collection (ATCC, USA) and cultured continuously in DMEM (Dulbecco's Modified Eagle Medium, supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin, Thermo Scientific) at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air. For cell uptakes study, RAW264.7 cells (1×105 cells/well) were placed in a flat bottom 24well plate in 500 μL culture medium ,100 μL

131I-PISO

(18.5 kBq/mL) was added and

incubated with the macrophage for different time (0, 5, 10, 30, 60, 90, 120 min). Cells were washed with 1 mL of PBS (pH = 7.4) for three times, after that, cells were lysed by addition of 500 μL NaOH (1 mol/L) for 5 min and radioactivity was counted using a 2480 Wizard2 Automatic Gamma Counter. For efflux study, RAW264.7 cells in 48 wells (1 × 105 cells/well) were divided into two groups (2  24 wells). Cells in experiment group were stimulated with LPS (500 ng/mL) for 6 h. Then the 131I-PISO (18.5 kBq/mL, 100 μL) was added in each well and incubated for 90 min. Cells were washed with 1 mL of PBS (pH = 7.4) for three times, followed by incubated in fresh 500 μL culture medium for different time (5, 10, 30, 60, 90 min). Then

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cells were washed with 1 mL of PBS (pH = 7.4) for three times and lysed by addition of 500 μL NaOH (1.0 mol/L) for 5 min. radioactivity was counted using a 2480 Wizard2 Automatic Gamma Counter. Metabolic Analysis of 131I-PISO in vivo 131I-PISO (37 MBq, 100 L) was injected intravenously through tail vein of the LPS treated

mice and normal mice (n = 3) in the metabolic study. Mice were sacrificed at 0.5, 1 h postinjection (p.i.), and the liver, plasma, and urine were collected. Then the liver was homogenized and extracted with 500 µL acetonitrile, followed by centrifuging at 12, 000 rpm for 5 min, passing through a 0.22 µm Millipore filter and analyzing the supernatants with a radio-HPLC. MicroSPECT/CT Imaging of O2•− Load In situ O2•− enrichment model mice (after 4 h intraperitoneal injection of xanthine/xanthine oxidase (X/XO)) and LPS-treated mice were administered with 7.4 MBq 131I-PISO through tail vein, followed by microSPECT/CT scans at 0.5 h, 1 h, and 2 h p.i., respectively. The imaging protocol was described in the supplemental methods section. MicroSPECT/CT Imaging of Inflammation The inflammatory model mice were obtained by administering complete Freund’s adjuvant. After 7 days, each mouse was administered intravenously with 18.5 MBq 125I-PISO (100 μL). Mice were anesthetized with 2% isoflurane and maintained 1~2% isoflurane

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throughout the SPECT/CT scanning session. The fluorescent imaging of inflammatory model mice was performed on a Carestream Fx Pro/FX imaging system. Mice were anesthetized with abdomen injection of 7% chloral hydrate solution (100 μL) and then subjected to imaging (Ex = 470 nm, Em = 535 nm, exposure time = 30 s) at 0, 5, 30 min, 1, 2, 5 h after intravenous (i.v.) injection of PISO (100 μL, 1.0 mg/mL in PBS 5% ethanol) for in vivo detection of inflammation. Biodistribution of Inflammatory Model Twenty inflamed mice (male, 18-20 g) were divided into five groups (n = 4) and injected with purified

131I-PISO

(0.37 MBq) through tail vein for each mouse. Then mice were

sacrificed at 2, 10, 30, 60, 120 min p.i., and interested organs were collected and weighted. Radioactivities in each organ were counted using a 2480 Wizard2 Automatic Gamma Counter. The uptakes in organs were expressed as the percentage of the injected dose per gram organ (%ID/g). Data and Statistical Analysis The regions of interest (ROIs) were drawn on the summed SPECT and fluorescent images manually. Relative fluorescence intensities and relative radioactivity counts were calculated by divide maximum ROI value. Quantitative radiotracer uptakes in vitro were calculated as counts per minute (CPM). Results are expressed as a mean ± standard deviation. The Pearson correlation coefficient was computed to assess the quality of linear

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correlations. Differences were considered significant if P values < 0.05.

Results Chemistry and Radiochemistry All the synthesized compounds were characterized by 1H-NMR, 13C-NMR and MS/HRMS respectively. The chemical purities of the precursor and the reference compound PISO were calculated as more than 95% from the radio-HPLC chromatograms (Supplemental Figure 1 and Figure 2A). Suggesting PISO is an acceptable reference standard for the corresponding radioactive tracers. Radioiodinated PISO was prepared from the tributyltin precursor (compound 3) via a radio-iododestannylation reaction within 40 min including HPLC purification. The overall radiochemical yields of 131/125I-PISO was 65.4 ± 9.2% (n = 6, end of synthesis, no decay corrected) with high radiochemical purities (> 98%) after radio-HPLC purification. The retention time of 131I-PISO and 125I-PISO were 8.49 min 8.42 min respectively, which were consistent well with the non-radioactivity reference compound PISO (8.10 min, Figure 2A, Supplemental Figure 2). The obviously different retention time makes the radiotracer could be separated well with the precursor (retention time 26.89 min). The specific activities of purified probes were estimated by radio-HPLC to be about 17.8 ± 2.2 (n = 3) GBq/μmol (131I-PISO) and 14.8 GBq/μmol (125I-PISO), respectively (Supplemental Figure 3 and Supplemental Table 1). 17



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Selectivity and Reactivity of PISO for O2·The fluorescence intensity generated from PISO after reacting with O2•− was about 200 ~ 500-fold higher than that of other substances, even these molecules with much higher concentrations than that of O2•−, which suggests that PISO exhibited excellent selectivity towards O2•− among the other ROS and substance (Figure 3A). The maximum fluorescence intensity was observed during pH 7 to 8, similar to the pH in the living body (Supplemental Figure 4). Additionally, superoxide dismutase as a specific scavenger of O2•− could inhibit fluorescence effectively in vivo (Supplemental Figure 5). Fluorescence intensity of PISA was found to increase with O2•− concentration linearly which means PISO could be used to reveal the concentration of O2•− quantitatively (Supplemental Figure 6). We further studied the kinetics of PISO in detection O2•− in vitro by HPLC analysis, and conversion reaction of PISO to PISA was finished completely within 1 min (Figure 2B). Stability and in vivo Conversion of 131I-PISO Over 98% of intact

131I-PISO

were identified after 60 min incubation in saline or serum

(Supplemental Figure 7). No metabolites appeared in HPLC analysis of the blood and urine (Figure 3B, samples were obtained from normal mice after 60 min i.v. injection of

131I-

PISO). About 51% 131I-PISO had been converted into 131I-PISA in the liver of LPS-treated mice due to the enriched O2•− induced by LPS, while only 10% conversion was detected in the liver of normal mice (Supplemental Figure 8). 18


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Partition Coefficient and Polarity Inversion of 131I-PISO The log P of 131I-PISO was 2.46 ± 0.11 (n = 3) compared with -1.63 ± 0.18 (n = 3) for 131IPISA (Figure 2C), which revealed the dramatic log P shift was due to the change of polarity. Furthermore, the retention time of PISO and PISA in reverse-phase HPLC analytical were 8.05 min and 3.02 min respectively (Figure 2B), which agreed well with their polarities. Cell Uptake and Efflux of 131I-PISO The uptake of

131I-PISO

in RAW264.7 cells increased with time and reached 27% at 90

min (Supplemental Figure 9) for the osmotic pressure in cell increase with the concentration and inhibit the permeation of more 131I-PISO. The efflux of 131I-PISO from cells in control group was much faster than that of LPS-treated cells with 59.2% in LPS and 41% in control at 90 min (Figure 2D), owning to polarity conversion and retention of the probe. MicroSPECT/CT Imaging of in situ Loaded O2·In situ O2•− enrichment model mice were prepared for SPECT/CT imaging study. In control, no noticeable signal appeared in mice abdomen (Figure 4A), indicating low background and non-specific signal accumulation. For X/XO treated mice the signal in abdomen increased significantly in O2•− enriched area at 2 h (Figure 4B). However, after i.p. injection with Tiron in the same spot to scavenge O2•−, the signal was eliminated in the X/XO injection spot (Figure 4C). After injection of 131I-PISO, whether in normal or X/XO treated 19



Page 20 of 34

mice, it shown a high uptake in liver. It might be due to its high lipophilicity (log P = 2.46). In blocked group the uptake didn’t decrease obviously, it suggested that the signal in liver were come from the compound converted

131I-PISA.

131I-PISO

To further correlate

and it has less effective blockage than that of 131I-PISO

uptake and O2•− local concentration

quantitatively, in situ model mice with graded O2•− induced by graded X/XO were created. As shown in the transversal SPECT/CT images (Figure 4D), there is a significant difference between mice mouse #1 and #2, and the two regions of mouse #3. And that the uptake of 9 μM xanthine regions was about 3-fold to the 3 μM xanthine regions (Figure 4E), which increased proportionally with the concentration of O2•−. MicroSPECT/CT Imaging of Endogenous O2•− MicroSPECT/CT imaging of the LPS treated mice was performed at 0.5, 1, 2 h after i.v. injection of 131I-PISO. At 0.5 h p.i., the radioactivity in the abdomen of control mice was similar to the low LPS dosage group, while obviously enhanced uptakes were monitored in the high LPS dosage group. After specific inhibition by Tiron, the radioactivity decreased obviously (Supplemental Figure 12). MicroSPECT/CT Imaging of Inflammation SPECT/CT imaging of inflammation model with

125I-PISO

were performed to detect the

endogenous O2•− during inflammation in vivo. As shown in Figure 5A, we observed a higher uptake of 125I-PISO in the inflamed ankle than healthy one. In fluorescent images, 20


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it showed a similar result with SPECT (Figure 5B). Both SPECT and fluorescent imaging correlated well with the observed feet swelling (Figure 5C). The time-activity-curves (TACs) of the selected regions derived from SPECT imaging during 180 min p.i. (Figure 5D) showed that 125I-PISO accumulated in the inflamed ankle quickly and reached a peak at 60 min. After that, it was cleared slowly from the inflamed ankle in the next 120 min. As a comparison, the uptake of

125I-PISO

in healthy ankles increased slowly, and the

radioactivities counts in inflamed ankles were about 2-fold to normal ankle. Biodistribution of 131I-PISO in Inflammation Biodistribution results in the inflammation model was shown in Supplemental Figure 15 and Supplemental Table 2. At 2 min p.i., most of the activity were accumulated in blood (12.74 ± 2.25 %ID/g), heart (10.26 ± 1.60 %ID/g), liver (17.93 ± 2.78 %ID/g) and lung (21.92 ± 1.96 %ID/g). The activities were washed out rapidly from the blood and lung with time (1.22 ± 0.11 %ID/g for blood, 5.44 ± 0.96 %ID/g for lung at 120 min p.i.). While uptakes in liver and spleen increase with time (24.27 ± 1.16 %ID/g in liver and 10.42 ± 1.50 %ID/g in spleen). The thyroid had moderate uptakes from 2 min to 120 min, indicating good stability of 131I-PISO in vivo. The uptake of inflamed ankle was higher than normal at

2

min

(2.15

±

0.14

vs.

1.69

±

0.09

%ID/g)

and

ratio

of

inflamed/normal peaked from 10 min to 30 min (inflamed uptake was about 2-fold to the non-inflamed), then uptake in the inflamed ankle decreased to normal ankle level at 120 21



Page 22 of 34

min.

Discussion Superoxide has been recognized as a vital signaling molecule in physiological and pathological processes in vivo 37-39. Although quite a few fluorescent and radioactive probes have been reported for ROS sensing, most of them were based on the oxidation strategy that suffers the interference of other reactive oxygen species. Inspired by the newly developed fluorescent probe (HKSOX-1) which was triggered by O2•– with unique strategy and outstanding sensitivity (estimated detection limit was about 23 nM)

31.

Hence we

designed and synthesized a analog of the reported probe and radiolabeled it for SPECT/CT imaging. Our probe (125/131I-PISO) should has similar sensitivity with its homologue compound HKSOX-1. The “polarity-inversing then trapped” strategy by negative charge and electrostatic interactions was adopted for dynamic capturing and signal accumulation of the relatively very short lifetime of O2•−. Fluorescent imaging alone works through the turn-on in fluorescence, which is limited for in vivo imaging and clinical translation for its insufficient penetration. After radiolabeling, better signal penetration and quantitation make the probe more promising for clinical use with gamma-ray detecting modalities such as PET or SPECT 40. The SPECT imaging probe 125/131I-PISO was prepared by a simple and efficient one-step labeling process less than 40 min including purification. About 500-fold enhancement in

22


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fluorescence intensity was observed toward O2•−, while PISO was inert toward common reactive species and substance except O2•− in vitro. Good performance of PISO under pH variations and fast enough reaction kinetics (< 1 min) also contribute to the feasibility of in vivo O2•− detection. Several experiments were further conducted to study the selectivity of

125/131I-PISO

in detection O2•− in vivo. SPECT/CT imaging of in situ O2•− enrichment

and blocked model mice were performed successfully with treated mice or LPS-treated mice, uptakes of

131I-PISO

131I-PISO.

With either X/XO

in O2•− enrichment regions were

higher than normal. The obviously blocked uptakes by Tiron again verified the specificity. Meanwhile, the uptakes calculated from SPECT images increased with the concentrations of O2•− proportionally. To diagnose the inflammatory diseases in their early stages requires a method that is sensitive and selective to detect and monitor the inflamed tissues

41-43.

SPECT/CT imaging observed inflamed paw sensitively. The TACs of

In this study,

125I-PISO

shown

that the maximum uptake in inflamed paws were about 2-fold to noninflamed paws, consistent with biodistribution results in inflammatory model. O2•− is not only a harmful agent causing oxidative damage in pathologies but also a vital signal molecule in the regulation of the metabolism in a variety of biological processes in vivo

44,45.

In neurodegenerative diseases and cancers, the homeostasis of ROS has been

destroyed for the production of ROS exceeds their catabolism. Over expressed ROS could

23



injury the proteins and DNA, then induce irreversible damage in vivo. Therefore, the O2•− could be a marker for the detection of neurodegenerative diseases and cancers in early stages. Besides, chemotherapy, radiotherapy, photodynamic therapy could produce O2•− for cell killing, and overdosing is prevailing to ensure the effectiveness. Hence, a useful tracer to selectively and quantitatively image either endogenous or exogenous O2•− in patients in real time will be very helpful in the clinic. Additionally, SPECT/CT imaging with our developed probe for the in vivo assessment of the efficacy of chemotherapy, radiotherapy, photodynamic therapy agents in the preclinical study should be a potent strategy for drug screening.

Conclusions Herein, we experimentally verified that SPECT/CT imaging with 125/131I-PISO might be a feasible method to revealing the biodistribution of O2•− in the whole bodies of living subjects and to gain insight into the relationship between ROS, pathological and diseases by non-invasive molecular imaging.

Competing interests The authors declare that they have no competing interests. Grant support: This study was financially supported by the National Natural Science Foundation of China (81501534, 81471707 and 81402648), National Key Basic Research 24


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Program of China (2014CB744503), Fourth Round Fujian Health Education Joint Research Projects (WKJ2016-2-08), Fujian Province Young Teacher Research Program (JA15010), Fundamental Research Funds for the Central Universities (20720180050) and Scientific Research Foundation of State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics (2016ZY002).

Acknowledgments We thank Dr. Liubin Feng from High-Field Nuclear Magnetic Resonance Research Center of Xiamen University for assistance in the NMR characterization. This work was funded by National Natural Science Foundation of China (81501534, 81471707 and 81402648), National Key Basic Research Program of China (2014CB744503), Fourth Round Fujian Health Education Joint Research Projects (No. WKJ2016-2-08), Fujian Province Young Teacher Research Program (JA15010), Fundamental Research Funds for the Central Universities (20720180050) and Scientific Research Foundation of State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics (2016ZY002). The authors declare no competing financial interest.

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For TOC only

29



Figure 1. Structure of 125/131I-PISO and the mechanism for O2•− detection.

30


Page 30 of 34

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Figure 2. (A) HPLC chromatogram of co-injection of PISO and

131I-PISO.

(B) Reaction

kinetics monitored by HPLC. (C) Dramatic shift of log P of 131I-PISO and 131I-PISA. (D) Slower efflux rates of

131I-PISO

in LPS stimulated macrophage compared with normal

macrophage. Error bars (C, D) represent one standard deviation (SD) of uncertainty (n = 3).

31



Figure 3. (A) Selectivity of PISO for superoxide. Fluorescence intensity generated by PISO (15 μM) reacted with various ROS, amino acids, metal ions, and other reductants for 1 h. Error bars represent one SD of uncertainty (n = 3). (B) Metabolic stability of 131I-PISO in vivo, (a, b) Representative HPLC profiles of the soluble fractions of blood samples at 0.5 h and 1 h p.i. (c, d) Representative HPLC profiles of the soluble fractions of urine samples at 0.5 h and 1 h p.i.

32


Page 32 of 34

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Figure 4. Semi-quantitative SPECT/CT of O2•− distribution with

131I-PISO.

(A) Normal

mice. (B) Endogenous O2•− induced by in situ (n = 3), with O2•− rich region clear illustrated in the red circles. (C) Tiron (a specific O2•− scavenger, 0.4 μg/g body weight) injected X/XO treated model mice (n = 3) as the blocked group. (D) Semi-quantitative SPECT/CT of O2•− distribution in vivo in model mice treated with X/XO at graded concentrations in the peritoneal cavity. (E) About 3-fold enhancement of 131I-PISO uptake derived from SPECT images was observed in 9 μM xanthine region compared to 3 μM regions. Error bars represent one SD of uncertainty (n = 3).

33



Figure 5. (A) SPECT/CT imaging of inflammatory model with 125I-PISO (18.5 MBq) after 1 h i.v. injection. (B) Fluorescence imaging of inflammatory model with PISO (1 mg/mL 100 μL) after 1 h i.v. injection. (C) Photo of the inflammatory model. (D) Time-activitycurves of 180 min SPECT scans focuses on the healthy and inflamed ankle. (E) Time-activity-curves of fluorescence intensity in the inflamed and healthy ankles in 300 min. Error bars (D, E) represent one SD of uncertainty (n = 3).

34


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A Highly Specific and Sensitive Radioiodinated Agent for in vivo

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