Dimethylformamide, sodium acetate, D-glucosamine hydrochloride, and N-methylmorpholine were purchased from Sigma-Aldrich (St. Louis, MO). 2-(p-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (p-SCN-Bn-DOTA) was purchased from Macrocyclics (Dallas, TX). Other commercially available chemicals were purchased from VWR International (San Diego, CA). All reagents were used as received. 18F-FDG was obtained from the Department of Nuclear Medicine at The University of Texas MD Anderson Cancer Center.
Synthesis of DOTA-DG
DG was reacted with p-SCN-Bn-DOTA in an aqueous solution at pH 7 for 12 hours at room temperature to produce DOTA-DG linked via a thiourea bond (). The progress of the reaction was monitored using high-performance liquid chromatography (HPLC). DOTA-DG was analyzed via electrospray ionization mass spectrometry using an Agilent LC/MSD TOF mass spectrometer (Agilent Technologies, Santa Clara, CA) equipped with a Vydac C-18 column (4.6×250.0 mm, 7-μm particle size, 300-Å pore size (Grace, Deerfield, IL). The HPLC eluting conditions were as follows: solvent: A, 0.1% trifluoroacetic acid (TFA) in water; B, 0.1% TFA in acetonitrile; gradient: B, 0-10% over 0-10 minutes; B,10-50% over 10-12 minutes; B, 50-80% over 12-20 minutes; B, 80% to 10% over 20-21 minutes. The flow rate was 1 mL/minute.
Synthesis of the precursor compound DOTA-DG and labeling of it with 68Ga. M.W., microwave heating.
Microwave heating-assisted 68Ga labeling of DOTA-DG
68GaCl3 in 0.1 N hydrochloric acid (370 MBq/0.3 mL) was obtained from 68Ge/68Ga generator (Eckert & Ziegler, Berlin, Germany). Two methods were used for labeling of DOTA-DG with 68Ga. In the conventional method, 370 MBq of 68GaCl3 in 0.3 mL of a 1-M aqueous solution of sodium acetate (pH 4) was added to a solution of DOTA-DG in distilled water (100 μg in 0.1 mL). The mixture was allowed to react at 90°C for 20 minutes in a water bath and then cooled to room temperature over 30 minutes. In the microwave heating-assisted method, the mixture of 370 MBq of 68GaCl3 in 0.3 mL of a 1-M sodium acetate solution and 100 μg of DOTADG in 0.1 mL of distilled water was heated in a microwave reaction apparatus (Discover; CEM Corporation, Matthews, NC) at a power setting of 25 W for 5 minutes and 50 W for an additional 5 minutes. The reaction mixture was then kept at 40°C for 5 minutes. DOTA was labeled with 68Ga similarly to DOTA-DG, and the resulting radiotracer, 68Ga-DOTA, was used as a control in biodistribution study.
Radiolabeled compounds were analyzed using HPLC. The specific activity of 68Ga-DOTA-DG was determined with a radio-HPLC chromatogram by dividing the integrated peak radioactivity of the radiotracer by its physical quantity derived from the corresponding ultraviolet absorbance and a calibration curve of known quantities of the unlabeled compound. The labeling yields were determined using instant thin-layer chromatography (ITLC) (German Science, Ann Arbor, MI) developed with a solution of 0.1 M citric acid in saline. The strips were scanned using an ITLC scanner (AR-2000; Bioscan, Washington, DC).
In vitro cell uptake of radiotracers
A431 human epithelial carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 in Dulbecco’s modified Eagle’s medium and Ham’s F12 nutrient mixture containing 10% fetal bovine serum (Gibco, Grand Island, NY).
Cell uptake assays were performed after seeding 1.2×106 A431 cells/mL/well in 24-multiwell culture plates. When the cells were about 80% confluent, each well was injected with 74 KBq of 68Ga-DOTA-DG or 18F-FDG in 1 mL of culture media (2 μg/mL for both radiotracers). A blocking study was performed with the addition of both radiotracers with 1 mg of cold deoxy-Dglucose to the wells (100 μL/well). After 60 minutes of incubation at 37°C, the culture media were removed, and the cells were washed twice with ice-cold Hank’s balanced salt solution (pH 7.3). The cells were then dissolved in 0.5% sodium dodecyl sulfate (0.5 mL/well). The radioactivity in the cells was measured using a gamma counter (Packard, Downers Grove, IL). The cell uptake of the radiotracers was calculated using the formula %uptake = (radioactivity of cells/total radioactivity) × 100%. This study was performed in triplicate.
The mice were kept under specific pathogen-free conditions and were handled and maintained according to Institutional Animal Care and Use Committee guidelines. A431 cells were inoculated subcutaneously into the right thighs of nude mice (20-25 g; Harlan Sprague Dawley, Indianapolis, IN) by injecting 1×106 viable tumor cells in a suspension of phosphate-buffered saline. When the resulting tumors grew to a diameter of 6-8 mm, the mice were allocated to three groups of three mice each. They were then injected intravenously with 68Ga-DOTA-DG or 18F-FDG (3.7 MBq/mouse). The animals were killed at 10, 30, and 60 minutes after injection. Blood, heart, liver, spleen, kidney, lung, stomach, intestine, muscle, bone, brain, and tumor tissues were removed, weighed, and counted for radioactivity using a gamma counter. Uptake of each radiotracer in various tissues was calculated as the percentage of the injected dose per gram of tissue (%ID/g).
A431 tumor cells were inoculated into nude mice as described above. When the resulting tumors grew to 6-8 mm in diameter, the mice were injected intravenously with 68Ga-DOTA-DG (20 μg, 14.8 MBq/mouse, 0.2 mL). The mice were then placed in the prone position for micro-PET (μPET) imaging. Prior to imaging, the mice were anesthetized with 2% isoflurane gas (Iso-Thesia, Rockville Centre, NY) in oxygen. During imaging, anesthesia in the mice was maintained with 0.5-1.5% isoflurane. μPET images were acquired 30-80 minutes after radiotracer injection using an R4 μPET scanner (Concorde Microsystems, Knoxville, TN).
Cell uptake and biodistribution data were analyzed using two-tailed, unpaired Student t-tests, with p values less than 0.05 considered to be statistically significant. The in vitro percentage of radiotracer uptake, in vivo percentage of injected radiotracer dose per gram of tissue, and tumor-to-nontumor ratios are presented as the mean ± standard deviation. All statistical computations were performed using the Excel software program (Microsoft Corporation, Redmond, WA).