Western blot analysis
According to the previously described methods (25,26), the expression of TSPO protein in RAW264.7 cell lines with or without LPS treatment is determined by western blot analysis. Shortly, RAW264.7 cells were lysed in 1 mL RIPA solution (containing 1% Triton X-100, 1% deoxycholate, and 0.1% SDS; Beyotime Biotechnology, Haimen, China) supplemented with a protease inhibitor (PMSF, Sangon, shanghai, China) for 30 min at 4 °C, then centrifuged at 12000 rpm for 30 min at 4 °C to obtain the total cell extracts. 15 mL cell extracts were subjected to SDS-PAGE and transferred onto a PDVF membranes by MiniPROTEAN (Bio-Rad, Hercules, California, USA). The PDVF membranes were blocked with TBST (10 mM pH 7.4 Tris-HCl, 150 mM NaCl, and 0.1% Tween-20) containing 5% skim milk for 2 h at 37 °C, and incubated overnight at 4 °C with anti-TSPO mAb (diluted 1:100). After washed with TBST three times, the membranes were again incubated with goat anti-mouse IgG labeled peroxidase (diluted 1:5000) for 1 h 37 °C. Protein of interest were visualized with ECL in Kodak Image Station 4000R (Carestream Health, Rochester, New York, USA). ß-actin was used as an internal control. All experiments were performed in duplicate.
Cellular immunofluorescence staining
Cellular immunofluorescence staining was performed as previously described (25,27). Briefly, the cell-seeded coverslips were washed and fixed. The TSPO mAb (diluted 1:100) and fluorescence (TRITC)-labeled secondary antibody (diluted 1:50; Gibco, Grand Island, New York, USA) were added restained with Hoechst 33258 (Beyotime Biotechnology, JiangSu, China). The sildes with cells were incubated without the TSPO mAb as control. The fluorescence images were captured at excitation laser of 360 nm and emission laser of 460 nm for Hoechst 33258, and at excitation laser of 488 nm and emission laser of 530 nm for FITC by using Olympus FV 1000 Inverted Confocal Fluorescence Microscope (Olympus, Columbia, South Carolina, USA). All experiments were performed in duplicate.
Cell viability assay
Cell viability was analyzed by MTT assay. RAW264.7 cells and 4T1 cells (with low TSPO expression) were seeded in 96 well plates at 1 × 104 cells and were treated with CB86 or CB86-DTPA suspensions (100 μL per well), respectively, at different concentrations (0, 1.25, 2.5, 5, 10, 20 μM in DMEM) for 24 hours at 37°C and 5% CO2. Subsequently, 10 μL of 5 mg/mL MTT was added to each well and incubated for an additional 4 h at 37 °C under 5% CO2. The optical density (OD) in each well was measured by a scientific microplate reader (Multiskan Spectrum; Thermo Fisher, USA). The OD at 490 nm was determined. The OD from the wells of the cells cultured with complete medium was taken as 100% viability. Relative cell viability (%) compared to control cells was calculated using the formula: % viability = OD (treated) / OD (control) x 100%.
Synthesis of coumarin-CB86
CB86 purity and molecular mass were determined by analytic scale reversed-phase high-performance liquid chromatography (HPLC, model: 3000 HPLC System, Dionex Corporation, Sunnyvale, California) and matrix-assisted laser desorption/ ionization–time of flight mass spectrometry (MALDI-TOF-MS, model: Perseptive Voyager-DE RP Biospectrometer, Framinghan, Massachusetts). After that, CB86 (50 mg) dissolved in DMSO (2 mL) and 7-Hydroxylcomuarin (1.62 g, 10 mmol) were added to ethanol (140 mL). The reaction mixture was stirred at 60 °C for 30 min and then was cooled to 5 °C in an ice bath. The byproduct was removed by filtration and then purified by column chromatography (hexane/ethyl acetate = 6:1, v/v) to give a white filtrate containing coumarin-CB86.
Fluorescence imaging of CB86 in living cells
The RAW264.7 cells stimulated with LPS were incubated in the probe coumarin-CB86 (25 μM) with or without CB86 (10.0 μg) for 2 h at 37 °C and washed with 0.1 M PBS (0.6 mL x 3) before observation. The cells were then stained for 10 min with MitoRed, a well-established mitochondrial dye. The cells were observed using the confocal fluorescence microscope (Olympus FV 1000 Inverted, Olympus, Columbia, South Carolina, USA) with a 63 × oil-immersion objective lens. The excitation wavelength was 346 nm, and emission was collected at 455±10 nm. All experiments were performed in duplicate.
Conjugation of DTPA-CB86
CB86 (50 mg) and DTPAA (200 mg) were dissolved in DMSO (2 mL) under vigorous stirring and stirred at room temperature for 24 h in the dark. The byproduct was removed by filtration to give a reddish-brown filtrate containing DTPA-CB86. The DTPA-CB86 was characterized by HPLC and MALDI-TOF-MS.
Labeling DTPA-CB86 with 99mTc
The radiolabeling method of DTPA-CB86 was performed as our previously described methods (28). The compound DTPA-CB86 was labeled with 99mTc using SnCl2·2H2O as a reducing agent. Briefly, 100 μg/100 μL of DTPA-CB86 and 20 μL of SnCl2 (2 mg/mL in 0.1 M HCl) were mixed in a vial. Next, 185~370 MBq of freshly Na99mTcO4 was added to the mixture. The reaction mixture was then incubated at 100 °C for 30 min to obtain the resulting radiotracer 99mTc-DTPA-CB86. The resulting solution of 99mTc-DTPA-CB86 was purified and analyzed by Sep-Pak C18 cartridge (GE Healthcare, Piscataway, New Jersey), and radio-HPLC (Thermo Scientific, Waltham, MA, USA). The mobile phase is presented below: A: H2O, B: 100% CH3OH; 0-10 min, B: 10%; 10-20 min, B: 90%; 20–30 min, B: 90%; 30-40 min, B: 10%; flow rate: 0.5 mL/min. The synthetic scheme of 99mTc- DTPA-CB86 is shown in Figure 1.
Determination of lipid-water partition coefficient of 99mTc-DTPA-CB86
To determine the hydrophilicity of 99mTc-DTPA-CB86, the partition coefficient (expressed as log P) was measured as our previously described methods (28). 200 μL 99mTc-DTPA-CB86 was added to 1 mL phosphate-buffered saline (PBS, pH = 7.4) saturated by n-octyl alcohol and 1 mL n-octyl alcohol saturated by PBS (pH = 7.4). After shaking for 5 min at room temperature, The solution was centrifuged at 3000 rpm for 5 min. Afterward, 100 μL of the organic phase and water phase were counted in a gamma counter, respectively. The averaged activities from each phase were used to calculate the log P values. The lipid-water partition coefficien (Po/w) of 99mTc-DTPA-CB86 was calculated as (cpm in organic phase)/(cpm in water phase). All the experiments were performed with triplicate samples and reported as mean ± SD.
In vitro stability analysis
In vitro stabilities in saline and mouse serum were determined similarly to the procedures previously described with minor modifications (29,30). 99mTc-DTPA-CB86 (5.55 MBq) in 250 μL of PBS was added to 2.0 mL of saline or mouse serum and was incubated at 37 °C for 1, 2, and 4 h. At each time point, the mixture in the mouse serum 1.85 MBq was precipitated with 300 µL of ethanol and centrifuged at 16,000g for 2 min. The supernatant was transferred to a new Eppendrof tube, and DMF (300 µL) was added to precipitated the residue of serum protein. After centrifugation, the supernatant or the mixture in saline was acidified with 300 µL of buffer A (water + 0.1% TFA) and filtered using a 0.2 -μm nylon Spin-X column (Corning Inc. Corning, New York). The filtrates were then analyzed by radio-HPLC under conditions identical to the ones used to analyze the original radiolabeled compound. The percentage of intact of 99mTc-DTPA-CB86 was determined by quantifying peaks corresponding to the intact and the degradation products. The assays were repeated twice.
Cell assays
Cell uptake, blocking, and efflux assays were performed as previously described with minor modifications (28-31). Briefly, the RAW264.7 cell lines were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. The cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C, with the medium changed every two days. A 70-80% confluent monolayer was detached by 0.1% trypsin and dissociated into a single cell suspension for further cell culture. All experiments were performed in duplicate.
Cell uptake assay The RAW264.7 cells were washed three times with 0.01 M PBS (pH 7.4) and dissociated with 0.25% trypsin-EDTA. DMEM medium was then added to neutralize trypsin-EDTA. Cells were spun down and re-suspended with serum free DMEM. Cells (0.5 × 106) were incubated at 37 °C for 30, 90, 180, and 240 min with 1.85 ×10-2 MBq 100 μL 99mTc-DTPA-CB86 in 0.5 mL serum-free DMEM medium. The non-specific binding of the probes with RAW264.7 cells was determined by co-incubation with 10.0 μg unlabeled DTPA-CB86. At each time point, after supernatants were removed, cells were washed with PBS and then lysed with 1 mL NaOH (1 M) for 5 min. The radioactivity of the lysates was measured using a gamma counter, and the cell uptake (counts/min) was normalized to the percentage of binding for analysis using Excel (Microsoft Software Inc., Redmond, Washington). All experiments were performed in duplicate.
Binding affinity assay The RAW264.7cells (0.2 × 106) were plated on 24-well plates one day before the experiment. After washing twice with DMEM, the cells were incubated at 25 °C for 3 h with 1.11 × 10-2 MBq 100μL 99mTc-DTPA-CB86 in 300 μL of DMEM with concentrations of unlabeled DTPA-CB86 ranging from 10-13 to 10-5 mol/L. After incubation, the cells were washed with cold PBS three times and detached with 1 mL NaOH (1 M) for 5 min. The radioactivity in the cells was measured using a gamma counter and were corrected for physical decay. The data were analyzed using GraphPad Prism (GraphPad Software Inc. San Diego, California), and the half maximal inhibitory concentration (IC50 value) of 99mTc-DTPA-CB86 was measured using a least squares fitting routine. All experiments were performed in duplicate.
Cell efflux study The RAW264.7 cells in separate 24 well plates were incubated with 1.11 × 10-2 MBq 100μL 99mTc-DTPA-CB86 at 37 °C for 240 min. After washing twice with PBS, cells were then incubated with culture medium for 30, 90, 180 and 240 min again to monitor the radioactivity efflux. At each time point, the cells were washed, lysed, and counted using a gamma counter. The cell retention rate of radioactivity was expressed as a percentage of the total input radioactive dose.
Induction of RA, assessment and anti-inflammatory therapy
Induction of RA. Experimental RA was induced in male Wistar rats according to the method previously described with some modifications (2,32). Briefly, the left ankle of each rat was injected with 0.1 mL of Complete Freund’s Adjuvant (CFA) with Mycobacterium butyricum 1% suspension in mineral oil.
Assessment of arthritis. The body weight of rats and development of RA disease were supervised daily by two observers. The severity of RA was evaluated according to the following scale: grade 1, detectable swelling in one joint; grade 2, swelling in two joints; grade 3, swelling in three joints; grade 4, severe swelling of the entire paw. The maximum score per animal for the four joints was 16. Each observation was done under short anesthesia using an isoflurane/oxygen mixture (2 to 3%). Each joint was graded, so the maximum score was 16 for a rat. Meanwhile, two weeks after injecting, the joint thickness was measured by a vernier caliper (Exploit Technology CO., LTD., Taiwan, China). Rats were allowed to grow to around grade 2 and 3 and then the RA rats were subject to in vivo biodistribution and imaging studies.
Anti-inflammatory therapy. RA rat with grade 4 (n =4 for each group) were administered with 0.5 mg/kg.d dexamethasone or saline by stomach perfusion once a day for 2 weeks, and then were imaged at the 7th day of and 15th day after treatment, respectively. The saline treated group as control group.
Biodistribution study
The animal procedures were performed according to a protocol approved by the Institutional Animal Care and Use Committee of Zhongshan Hospital Xiamen University. RA rats (n =4 for each group) were injected with 99mTc-DTPA-CB86 (0.37 MBq, 100μL) through the tail vein. At 30, 90, and 180 min after injection, the mice were sacrificed, and the left inflammatory ankles and normal tissues of interest were removed and weighed, and their radioactivity was measured in a gamma counter. The radioactivity uptake in the left inflammatory ankles and normal tissues were expressed as a percentage of the injected radioactivity per gram of tissue (%ID/g). In order to study the in vivo TSPO targeting specificity of 99mTc-DTPA-CB86 based on the previous studies (28,29), unlabeled DTPA-CB86 (300 μg) was co-injected with 99mTc-DTPA-CB86 in RA rats (n =3 for each group) via a tail vein, and biodistribution studies were conducted at 180 min after injection. The radioactivity ratios of the left inflammatory ankle to blood (LIA/B) and the left inflammatory ankle to muscle (LIA/M) were calculated.
SPECT imaging
SPECT imaging of RA rats was performed using a nanoscan SPECT/CT preclinical imager (Mediso, Hungary). The RA rats (n =4 for each group) were injected with 99mTc-DTPA-CB86 (0.37 MBq, 100μl) with or without co-injection of unlabeled DTPA-CB86 (300 μg) through the tail vein. At 30, 90, and 180 min after injection, the mice were anesthetized with 2% isoflurane and placed on SPECT bed (ventral side down). SPECT acquiring parameters were as follows: 140 keV energy peak for 99mTc, window width of 20%, matrix of 256 × 256, medium zoom, and frame: 30 s. Whole body static images (200,000 counts) were acquired with a matric of 218 × 218, and zoom of 2.0. CT data were acquired using an X-ray voltage biased to 50 kVp with a 670 μA anode current, and the projections were 7200. Regions of interest (ROIs) were drawn over the left inflammatory ankle and normal muscle, and then the ratios of the left inflammatory ankle to muscle ((LIA/M) were calculated.
Histological evaluation of the RA model
The left inflammatory ankles, contralateral normal ankles from RA rats were harvested and immediately frozen in dry ice. Thawed tissues were sliced into pieces. Hematoxylin and Eosin (HE staining), and immunohistochemistry (IHC) tests were performed on 5 μm frozen sections of ankle slices.
HE staining was conducted according to routine protocols. Briefly, after deparaffinization and rehydration, 5 𝜇m longitudinal sections were stained with hematoxylin solution for 5 min followed by 5 dips in 1% acid ethanol (1% HCl in 70% ethanol) and then rinsed in distilled water. Then the sections were stained with eosin solution for 3min and followed by dehydration with graded alcohol and clearing in xylene. The mounted slides were then examined and photographed using an Olympus BX53 fluorescence microscope (Tokyo, Japan).
Immunohistochemical staining
The slides were blocked with 5% goat serum in PBS containing 0.1% Triton X-100 for 1 h at room temperature and then incubated with rabbit anti-rat TSPO (1:200, Sigma) antibodies for 2 h at room temperature. After being washed, the slides were then incubated for 2 h at room temperature with secondary antibodies Goat anti-mouse IgG antibody (1:1,000; Sigma).The slides were then examined and photographed using an Olympus BX53 fluorescence microscope (Tokyo, Japan).
Statistical methods
The experimental data were analyzed by SPSS 18.0 (SPSS Company, Chicago, IL, USA). Statistical analysis was performed using two tailed Student's t-test for unpaired data. Data are expressed as mean ± standard deviation and P < 0.05 was considered to indicate a statistically significant difference.