2.1 Materials

All chemicals were obtained commercially and used without further purification. Methyl trifluoromethane- sulfonate was purchased from Matrix Scientific. Ethyl 4-dimethylaminobenzoate was purchased from J & K Chemical LTD. Tetrapropylammonium hydroxide solution (1mol/L in water) was purchased from Aladdin Chemistry Co., Ltd. N, N, N′, N′-Tetramethyl- O-(N-succinimidyl) uranium tetrafluoroborate (TSTU) was purchased from Shanghai Medpep Co., Ltd. Ethyl 4-(trimethylammonium triflate) benzoate and unlabelled N-succinimidyl 4-fluorobenzoate (SFB) were synthesized according to the method of Haka et al.^[17] and Johnstrom et al.^[18], respectively. Galactosyl- neoglycoalbumin (NGA) was prepared by the procedures of Zhang et al.^[6], with 25 galactose units attached to each molecule.

The analytical HPLC system included a binary HPLC pump (Waters 1525, USA), a UV detector (Waters 2487, USA) and a flow scintillation analyzer (Radiomatic 610TR, Perkin-Elmer, USA). Reversed- phase C-18 column (4.6× 250 mm, 5 μm particle size, Jiangsu Hanbon Science & Technology Co., Ltd) was eluted at a flow rate of 1mL/min as described in the experimental part. The absorbance was monitored at 254 nm. The TSKgel G2000SW_XL column (7.8×300 mm) was purchased from TOSOH Corp., Japan. The Sep-Pak Light Waters Accell Plus QMA cartridge (Waters, USA) was activated with NaHCO₃ and water before use. Reversed-phase extraction Sep-Pak C18 Plus cartridges (Waters, USA) were activated with methanol and water before use. A PD-10 column (filled with Sephadex G25) was purchased from GE Healthcare. The ICR mice were provided by the Comparative Medicine Center of Yangzhou University.

2.2 Synthesis of 4-¹⁸F-fluorobenzoic acid, ¹⁸F-FBA

Automated synthesis of ¹⁸F-labeling was carried out on an ¹⁸F-multifuntion synthesizer (Beijing PET Technology Co. Ltd., PRC) with a computer interface. The ¹⁸F-F^– was transferred in a pneumatic transport system from the cyclotron to the radiopharmaceutical laboratory. The ¹⁸F-F^– was separated from the ¹⁸O– H₂O using an anion exchange cartridge (Sep-Pak Light Waters Accell Plus QMA cartridge). The synthesis program was started with the elution of ¹⁸F-fluoride from the anion exchange cartridge into the reactor 1 using a solution of Kryprofix 222 (19.5 mg) and potassium carbonate (4.5 mg) in aqueous MeCN (1.5 mL, 90% MeCN) from vial 1 into the reaction vessel 1. The reaction vessel 1 was heated at 116°C and the solvent evaporated with a stream of nitrogen. The anhydrous MeCN (2 mL) was added from vial 2 and evaporated to dryness at 116°C for about 5 min. The solution of ethyl 4-(trimethylammoniumtriflate) benzoate 1 (10 mg) in anhydrous MeCN (1mL) was added from vial 3 to the ¹⁸F-KF-K222 complex and the mixture was heated five times to 90°C for 10 min with 5 s mixing between heating periods. The 0.5 mL NaOH (0.5 M) was delivered from vial 4 and heated three times to 90°C for 5 min with 5 s mixing between heating periods. After cooling for 2 min and acidifying with 0.1mol/L HCl (5 mL, vial 5), the solution was loaded onto a Sep-Pak C₁₈ cartridge. The cartridge was dried using nitrogen and the product was eluted with MeCN (3 mL, vial 6) into a second reaction vessel.

2.3 Synthesis of N-succinimidyl-4- ¹⁸F-fluoro- benzoate, ¹⁸F-SFB

The 4-¹⁸F-fluorobenzoic acid was eluted from the Sep -Pak C18 cartridge directy to the reaction vessel 2, and the 40 μL tetrapropylammonium hydroxide solution (1 mol/L in water) was added in the second reaction vessel previously. The mixture was heated at 116°C and evaporated to dryness. After cooling, a solution of TSTU (12 mg) in anhydrous MeCN (0.8 mL) was delivered to the reaction vessel from vial 11 and the mixture was heated three times to 90°C for 5 min with 5 s mixing between heating periods. After cooling for 2 min and acidified with 0.1mol/L HCl (5 mL, vial 12), the solution was loaded onto a Sep-Pak C₁₈ cartridge. Finally, the ¹⁸F-SFB was eluted with 3 mL MeCN (vial 13). The product was analyzed using radio-HPLC and radiochemical purity was evaluated.

2.4 Conjugation of ¹⁸F-SFB to NGA

The MeCN solution of ¹⁸F-SFB was evaporated to dryness with a stream of nitrogen. The residue was redissolved with 50 μL MeCN. The solution of NGA (1 mg, 200 μL, 0.014 μmol) in 0.1 mol/L borate buffer (pH 9.0) was added and reacted for 30 min at room temperature. The crude product was loaded onto a PD -10 column, eluted with 0.05 mol/L phosphate buffer (pH 7.5). After purification, the radiochemical purity was evaluated by radio-HPLC.

2.5 Radiochemical analysis

Analytical HPLC was performed using a Lichrospher C18 reversed-phase column (4.6×250 mm, 5 μm). The column effluent was monitored using a UV detector (Waters 2487, USA) and a flow scintillation analyzer (Radiomatic 610TR, Perkin-Elmer, USA) for radio- activity. ¹⁸F-SFB was analyzed using the solution of H₂O and MeCN (v/v =55:45) with 0.1% trifluoro -acetic acid (TFA) as mobile phase at flow of 1ml/min; UV (λ=254 nm).

¹⁸F-FB-NGA was analyzed with a TSK-GEL G2000SW_XL column (7.8×300 mm) using 0.1 mol/L PB, 0.9% NaCl and 0.05% NaN₃ as mobile phase at flow of 1.0 mL/min; UV (λ=280 nm). ¹⁸F-FB-NGA was incubated at room temperature for 4 h. The radiochemical purity (RCP) was evaluated by radio- HPLC at every single hour.

2.6 Biodistribution

Biodistribution of ¹⁸F-FB-NGA in normal mice was studied. ¹⁸F-FB-NGA (200 μL, about 0.37 MBq contained about 3 μg NGA) was injected by the tail vein. At the selected time points (10, 30 and 60 min), mice (n=5 at each time point) were sacrificed, and major organs and tissues were collected and weighed. The radioactivity in these tissues was measured using a gamma counter (1470 Automatic Gamma Counter, Perkin-Elmer, USA). The results were presented as the percentage injected dose per gram of tissue (% ID/g). For each mouse, the radioactivity of tissue samples was calibrated against a known aliquot of the injected activity. The mean uptake (% ID/g) for each group of animals was calculated with standard deviations.

2.7 MicroPET imaging

PET scans and image analysis were performed using a microPET Inveon rodent model scanner (Siemens Medical Solutions USA, Inc.). The normal and the blocking mice were tail-vein injected with about 200 μL of ¹⁸F-FB-NGA (about 3.7 MBq contained about 30 μg NGA) under isoflurane anesthesia and subjected to a 30-min dynamic scan starting after injection. The images were reconstructed by 3-dimensional ordered- subsets expectation maximization (OSEM) algorithm. The blocking mouse was pre-injected with 0.2-mL NGA (3 mg) 30-min ago.

3.1 Radiosynthesis

¹⁸F-fluorination of NGA was performed using ¹⁸F-SFB (Scheme 1). ¹⁸F-SFB was synthesized inside the fully automatic multifunction module with a decay- corrected radiochemical yield of 42.7 ± 5.9% (n = 8) in 55–65 min. The radiochemical purity of ¹⁸F- SFB was greater than 98%, as determined by analytic HPLC. Starting with ¹⁸F-F^–, the total radiolabeling time of ¹⁸F-FB-NGA took 120±10 min, including final purification. Labeled with 185–1850 MBq ¹⁸F-SFB, the specific activity of ¹⁸F-FB-NGA was estimated to be 7.83–78.3 TBq/mmoL. After purifying with PD-10 column, the radio chemical purity of ¹⁸F-FB-NGA was above 98% as determined by radio-HPLC. ¹⁸F-FB- NGA is stable for up to 4 h. The overall radiochemical yield with decay correction was 25–30%.

Yields for the conjugation correlate with several reaction parameters, such as the pH value of the reaction mixture and the initial protein concentration. As shown in Table 1, the radiochemical yield was dependent on the pH value. NGA was labeled with ¹⁸F by coupling with ¹⁸F-SFB under a basic condition. The percentage yield of ¹⁸F-FB-NGA gave a best labeling yield by the pH= 9 at 30 min. The radiochemical yield of ¹⁸F-FB-NGA increased from 4.3% to 58.1% with the concentrations of NGA from 0.1 to 5.0 mg/mL at pH=9 (Table 2). We obtained lower radiochemical yields when the concentration of NGA is less than 5 mg/mL.

Starting with ¹⁸F-F^–, the ¹⁸F-SFB was synthesized by three-step reaction. The NGA was reacted with activated ester ¹⁸F-SFB through ε-amino groups of lysine residues under basic condition. The coupling yield for NGA is relatively lower than HSA. Because many ε-amino groups of lysine of NGA have been coupled with galactose groups, its coupling with ¹⁸F-SFB is lower than that of HSA.

3.2 Radiochemical analysis

The HPLC chromatograms of ¹⁸F-SFB and ¹⁹F-SFB were presented in Fig.1. The resulting ¹⁸F-SFB was determined with a radiochemical purity of 98.2% at the same retention time as ¹⁹F-SFB. The retention time of ¹⁸F-SFB in our gradient system was 10.4 min.

Fig.1Full-screen View

The chromatograms of the purified ¹⁸F-SFB characterized by a radioactivity detector and ¹⁹F-SFB by a UV detector (254 nm) in the same analytical HPLC condition. The resulting ¹⁸F-SFB was determined with a radiochemical purity of 98.2% at the same retention time as ¹⁹F-SFB.

Fig.2 shows the HPLC characterization profile of the ¹⁸F-FB-NGA and NGA. The resulting ¹⁸F-FB-NGA was determined with a radiochemical purity of 98.6% at the same retention time as NGA. The retention time of ¹⁸F-FB-NGA was 6.8 min.

Fig.2Full-screen View

The chromatograms of purified ¹⁸F-FB-NGA characterized using a radioactivity detector and NGA using a UV detector (280 nm) in the same HPLC condition. The resulting ¹⁸F-FB-NGA was determined with a radiochemical purity of 98.6% at the same retention time as NGA.

3.3 Biodistribution

To evaluate tissue distribution characteristics of ¹⁸F- FB-NGA, we performed a biodistribution experiment using ICR mice. The data expressed as the percentage administered activity (injected dose) per gram of tissue (%ID/g) (Table 3). ¹⁸F-FB-NGA showed well liver accumulation. The liver uptake was 39.47 ± 3.42 at 10 min post-injection time; and 12.12 ± 6.11, at 30 min; and 5.39 ± 0.92%ID/g, at 60 mi. While the kidney uptakes were 11.19 ± 6.28 and 29.96 ± 4.27%ID/g at 10 and 30 min post-injection. The radioactivity in blood decreased rapidly. At 10 min after injection, the radioactivity concentration in blood was only 1.69 ± 0.21%ID/g. The results of biodistribution indicate that the ¹⁸F-FB-NGA has high affinity with the ASGP receptor and may get a quality imaging. It makes possible to diagnose liver disease by a non-invasive method. ¹⁸F-FB-NGA could be used as a receptor- specific radiopharmaceutical with potential applications in liver imaging for the evaluation of hepatocytic function.

3.4 MicroPET imaging

Dynamic microPET scans were performed on the normal and block groups, and the selected coronal images at different time points after injection of ¹⁸F- FB-NGA are shown in Fig.3. The normal and block groups were significantly different, especially in the heart and liver. The normal group showed low cardiac uptake and high liver uptake at 5 min after injection. The liver was clearly visible with high contrast to background, and there were no significant uptakes in other organs in the abdomen at 10 min after injection. After 30 min, the outline of liver was still clear.

Fig.3Full-screen View

Coronal microPET images of mice. The upper is the normal group images at 5, 15, and 30 min after injection of ¹⁸F-FB-NGA (3.7 MBq). The lower is blocking group images at 5, 15, and 30 min after injection of ¹⁸F-FB-NGA (3.7 MBq) after using free NGA as blocking agent (3 mg per mouse).

On the contrary, the liver accumulation was significant lower after blocking, and the outline of liver was vague. There was no significant uptake in the liver after injection in the block group. The block group showed high cardiac uptake during the 30 min. The microPET imaging studies with the normal and the block groups clearly showed the ASGP receptor- targeting avidity and specificity of the imaging agent ¹⁸F-FB-NGA in vivo.

The microPET of ¹⁸F-FB-NGA was evaluated with high liver accumulation and certain retention. The liver uptake was decreased significantly with free NGA after blocking. The block group showed high cardiac uptake. The result of microPET coincided with biodistribution. Both of the experiments showed high affinity of ¹⁸F-FB-NGA with the ASGP receptor.