All experiments were conducted following the relevant ethical and safety guidelines. Studies were conducted under the supervision of the MD Anderson Institutional Animal Care and Use Committee (protocol no. 00001179). Radiation handling was conducted under the supervision of the MD Anderson Radiation Safety Committee (authorized user protocol 1446).
Reagents and instrumentation
Reagents and solvents were purchased from Thermo Fisher Scientific and Sigma-Aldrich, unless otherwise specified, and used without further purification. 18F, as an aqueous solution, and [68Ga]GaCl3 were purchased from MD Anderson’s Center for Advanced Biomedical Imaging Cyclotron Radiochemical Facility (CRF). Radioactivity was measured using a dose calibrator (Capintec, CRC-15R). 1H and 13C NMR spectroscopy was performed at MD Anderson Cancer Center on a 500-MHz or 600-MHz spectrometer (Bruker) using tetramethylsilane as an internal reference. Mass spectronomy was performed at MD Anderson Cancer Center on a liquid chromatography mass spectrometer with an ACQUITY TQ Detector (Waters) using electrospray ionization.
Synthesis of precursor 2
Commercially available 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-ol (ChemBridge) (1, 100 mg, 0.37 mmol) was dissolved in dichloromethane (1.5 ml), and Boc2O (81 mg, 0.37 mmol) and DMAP (45 mg, 0.37 mmol) were added in turn. The mixture was stirred at room temperature overnight; the solvent was removed; and the crude residue was purified on silica gel (Biotage, % AcOEt in hexanes: 0% for 4 cv, 0%→20% in 5 cv, 20% for 2 cv). Protected naphthol 2 (110 mg) was obtained as a white solid in 80% yield (Extended Data Fig. 1).
2: 1H NMR (500 MHz, chloroform-d) δ 8.84 – 8.76 (m, 1H), 8.08 (d, J = 7.6 Hz, 1H), 8.00 (dt, J = 8.1, 1.0 Hz, 1H), 7.58 –7.49 (m, 2H), 7.32 (d, J = 7.6 Hz, 1H), 1.58 (s, 9H), 1.41 (s, 12H). 13C NMR (126 MHz, CDCl3) δ 151.6 (s), 149. 6 (s), 138.4 (s), 135.6 (d), 128.6 (d), 126.9 (d), 126.6 (d), 126.0 (s), 121.2 (d), 116.9 (d), 83.8 (s), 83.7 (s), 81.0 (s), 27.8 (q, 3 C), 24.9 (q, 4 C). MS (ESI) m/z (% relative intensity) 271 (80), 393 [M + Na]+ (40).
Synthesis of [18F]4FN
Radiosynthesis of [18F]4FN was performed on a TRACERlab FX FN automatic module (GE Healthcare). Aqueous [18F]fluoride was adsorbed on an ion exchange cartridge (pre-conditioned Sep-PAK Light QMA Cartridge, ABX). [18F]Fluoride was flushed into the reaction vial with a potassium carbonate and Kryptofix 222 water/CH3CN solution (700 μl; 52.8 mg of K2CO3, 240.1 mg of K222, 4 ml of water, 16 ml of CH3CN). The solution was dried under vacuum and nitrogen flow at 60 °C for 2 min. Dry CH3CN (500 μl) was added, and the mixture was azeotropically dried at 120 °C for an additional 3 min. Synthesis of 4-[18F]fluoro-1-naphthol was carried out by adding Boc naphthol boronate 2 (7 mg) and CuOTf2(Py)4 (15 mg, Sigma-Aldrich) in dry DMF (600 µl) to the dried [18F]fluoride. The mixture was stirred at 110 °C for 20 min and cooled down to 80 °C, and HCl 1 M (1 ml) was added. The mixture was stirred at 80 °C for 10 min, cooled down to 30 °C and diluted with water (3 ml). Crude was purified by semi-preparative HPLC (Luna 5-µm C18(2) column, 100 Å, 250 ×10 mm) eluting with a 50% MeCN/water (0.085% H3PO4) mobile phase (room temperature, 15 min). Radioactive product was collected into the TRACERlab collection flask pre-filled with water (50 ml). Solution was loaded onto two in-series tC2 cartridges (Sep-PAK, Waters). Cartridges were washed with 6 ml of water, dried under nitrogen and eluted with ethanol. The overall synthesis time was approximately 70 min. Activity was determined with a dose calibrator (Capintec, Mirion Technology), and a sample was taken for quality control. Quality control was performed by analytical radio-HPLC (Agilent 1260 Infinity II) using a Waters XBridge C18 column (3.5 µm, 4.6 × 250 mm; Method: A-water (0.1% TFA), B-MeCN (0.1% TFA) B: 40% for 3 min, 40–95% in 7 min, 95% for 1 min, flow 1 ml min−1). The identity of the radiolabeled compound was confirmed by co-elution with 4-fluoro-1-naphthol (Toronto Research Chemicals) (Extended Data Fig. 1).
[18F]4FN stability testing
For shelf stability, [18F]4FN, in its final formulation of 10% ethanol/PBS, was tested for stability every hour, up to 4 h after formulation. [18F]4FN was maintained in an open-air, non-inert environment during the testing period. For plasma stability, [18F]4FN was incubated in mouse plasma at 37 °C for 1 h. Samples were analyzed by analytical radio-HPLC (Agilent 1260 Infinity II) using a Waters XBridge C18 column (3.5 µm, 4.6 × 250 mm; Method: A-water (0.1% TFA), B-MeCN (0.1% TFA), B: 40% for 3 min, 40–95% in 7 min, 95% for 1 min, flow 1 ml min−1).
Synthesis of 68Ga-citrate and [18F]FDG
68Ga-citrate was synthesized following a literature procedure59. [18F]FDG was obtained from the CRF.
In vitro enzyme assays
In 1.5-ml autoclaved plastic tubes (Eppendorf), [18F]4FN was incubated with 0.2 µg of recombinant human MPO (Millipore), H2O2 (100 µM) or both in 100 µM PBS (Sigma-Aldrich). [18F]4FN was incubated at 37 °C for 1 h. Reactions were quenched with 100 µl of ice-cold ethanol and maintained on ice until radio-HPLC analysis. In parallel, 100 µM L-012 (Wako) was reacted with the same reagents on the same day in black-walled 96-well plates (Corning) to validate activity and specificity.
In cellulo radiotracer assays
Human HL-60 acute promyelocytic leukemia cells (American Type Culture Collection, CCL-240) were cultured in IMDM with 20% FBS. The HL-60 cells were differentiated into a neutrophil-like phenotype by incubation with 1 μM all-trans retinoic acid (Sigma-Aldrich) in culture medium for 3 d before the assay. Cells were transferred from culture medium to 0.6-ml tubes containing 1 million cells in 0.5 ml of MEBSS with 1% bovine calf serum on the day of the assay. The tubes were incubated at 37 °C for 1 h. Next, 20 μM 4-ABAH (Cayman Chemical), 10 μM diphenyleneiodonium chloride (Selleck Chemicals) or vehicle was added to the tubes, which were allowed to incubate for 30 min at 37 °C. The cells were then activated with 1 μM PMA (Sigma-Aldrich) or treated with vehicle and incubated for an additional 30 min at 37 °C. All tubes were then incubated with 0.169 ± 0.007 μCi of [18F]4FN for 30 min at 37 °C to allow cell uptake and retention. Tracer retention was stopped by adding 25 μl of 6.1 N trichloroacetic acid to the tubes on ice. The cells were centrifuged at 1,200 r.p.m. for 3 min at 4 °C, and the extracellular space was sampled for activity counting. Cell pellets were washed with ice-cold MEBSS containing 1% bovine calf serum and re-suspended in new tubes before centrifuging and washing again. Sodium borate buffer (10 mM) containing 1% SDS was added to cell pellets to allow lysis at room temperature for 30 min. Cell lysates were sampled for activity counting, and the remaining lysate was allowed to decay before a BCA assay to obtain protein concentration. Cell-associated and extracellular counts were obtained using a gamma counter (2480 WIZARD2 Automatic, PerkinElmer). Data were quantified as the ratio of cell-associated counts per milligram of protein over concentration of radiotracer in the extracellular space.
PET/CT imaging and analysis
For [18F]FDG PET/CT imaging experiments, mice were fasted for more than 4 h before injection. Mice were injected i.v. under light anesthesia with [18F]FDG (obtained from the CRF at MD Anderson) and then allowed to awaken, move and drink water ad libitum. Similarly, but without fasting, mice were injected i.v. (unless noted) with [18F]4FN. Cages were kept on a cage warmer during tracer distribution and washout periods. Mice were scanned at the indicated times after injection with a 10-min PET/CT acquisition (Albira, Bruker) using a 15-cm field of view (FOV); CT images were acquired for fusion using a 7-cm FOV, stepped and stitched if required. PET reconstruction was by MLEM 3D, 12 iterations, with decay, random and scatter correction applied to 500-μm isotropic voxels. CT is reconstructed with filtered back projection and 500-μm isotropic voxels. The preclinical PET system at the MD Anderson Small Animal Imaging Facility includes daily quality control with flood phantoms as well as at least one annual preventive maintenance, including quantitative calibration. Actual injected dose was calculated based on measuring the pre- and post-injection activity in the syringe with a dose calibrator (Capintec), and mice were individually weighed for static images. Image data were decay-corrected to injection time (Albira, Bruker) and corrected for individual injected dose (%ID/cc) and, where indicted, normalized to animal weight, expressed as SUV (g/cc) (PMOD, PMOD Technologies). For dynamic images, mice were injected on the imaging system, and %ID/cc was calculated on a per-mouse basis from the image-derived whole mouse activity.
Inflammation models in vivo
Whole-body inflammation was induced in female C57Bl6/N mice with LPS (Escherichia coli 055:B5 Ultrapure, InvivoGen) injection, 20 mg kg−1 i.p., and compared to saline control. Four hours later, mice were imaged with [18F]4FN or 68Ga-citrate as above and euthanized before onset of major symptoms.
Acute arthritis was induced with intra-articular LPS (20 µg in 20 µl of saline) into female C57Bl6/N (Taconic) mice; saline injection served as a control. Twenty-four hours later, mice with visibly inflamed ankles were injected with [18F]4FN i.v. and scanned continuously for 1 h with PET in list mode. Images were reconstructed, and PET/CT TACs were quantified. On the same day, mice were injected with L-012 sodium salt in saline (20 mg kg−1 i.p.) and imaged in bioluminescence and reflectance mode 10 min after injection on a bioluminescence/X-ray imaging system (Xtreme, Bruker). Images were acquired with a 19-cm FOV, f/1.1, binning 4 × 4, 243 s or 4 min. Mice were then euthanized, and ankles were harvested, fixed, decalcified and stained with H&E (Histochemistry Core, MD Anderson).
To induce acute contact dermatitis, adult female C57Bl6/N mice (Taconic) or female BALB/cN mice (BALB/cAnNTac, Taconic) (20–30 weeks old) were topically treated with PMA (Sigma-Aldrich, 400 µM, ethanol) on the right ear or vehicle (ethanol) on the left ear. Twenty-four hours later, mice were injected with [18F]4FN or [18F]FDG as indicated and imaged.
Female Cybb−/− mice (also known as Nox2−/−) and Mpo−/− mice were obtained from Jackson Laboratories and compared to strain- and age-matched C57Bl6/J mice, also from Jackson Laboratories.
Reporting Summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
