RPMI-1640 media were purchased from LONZA (MD, Walkersville, USA). DyLight 488 NHS Ester was purchased from Thermo Scientific (NH, Hudson, USA). Fetal bovine serum (FBS), penicillin, streptomycin, L-glutamine, 0.25% trypsin-EDTA, dimethyl sulfoxide (DMSO), phosphate-buffing saline, formaldehyde (CH2O), sucrose, propidium iodide and crystal violet dyes were purchased from Sigma-Aldrich Co., Ltd., Louis St.
AR42J pancreatic cancer cells were purchased from ATCC (Manassas, VA, USA) and cultured in RPMI-1640, supplemented on a 100 mm plate with 100 units/mL penicillin, 100 g/mL streptomycin and 10% fetal bovine serum. In addition, AR42J pancreatic cancer cells were cultured at 37℃ for fusion of up to 90% in a 5% CO2 atmosphere in air.
Male C57BL/6 mice (18 - 22 g, 6 weeks) were supplied by Nara Biotec Inc. (Seoul, Korea). All animals were kept in wire cages at 20 and 22 ℃ and given standard laboratory chow and water ad libitum, and the humidity was maintained at 50 ± 10 %. The management protocols and test system and animal care were authorized by the committee of Korea Atomic Energy Research Institute. All experiments were conducted in accordance with the relevant guidelines and regulations.
The DOTA-[Nle]-cCCK was synthesized by the automated Multiple Biomolecular Synthesizing System from Peptron, Daejeon, South Korea (Fig. 1). Fmoc-amino acid bond 4-Methylbenzhydrylamine (MBHA) resin was used as a fixed polymer support for solid phase synthesis. The Fmoc protector was removed from the resin boundary Fmoc-Met-OH under standard cutting conditions, and as a result, the peptide was separated from the polymer support by mixing with 90% TFA containing 2.5% triisopropylsilane (TIS), 2.5% ethianisol (EDT), 2.5% thionisol and 2.5% deionized water .
2.5 Combining receptors
AR42J cells were cultured in cover glass-bottom plate for the analysis of binding a residual amount of DOTA-[Nle]-cCCK. AR42J cells were treated for 20 minutes with 10 mL of DyLight 488 NHS Ester or DyLight 488 NHS Ester-conjugated DOTA-[Nle]-cCCK, and propidium iodide was added for nuclear detection. The cells were fixed with 4% CH2O and 3% sucrose. Fluorescent microscopy (Leica Microsystems, Wetzlar, Germany) was used to observe the stained cells.
DOTA-[Nle]-cCCK was liquefied with a sodium acetate buffer (50 mM, pH 5.5) to give 10-6 mol/mL. The final volume was 1 mL by injecting a diluted 44ScCl3 or 47ScCl3 solution (37 MBq) of 5.5M HCl into the vial of the peptide (10-8 mol) solution. Then, the bottle was heated at 90°C for 20 minutes. Radioactive labeling yield, radioactive chemical purity, and stability of the radioactive labeling compounds were analyzed by a water chromatograph equipped with an X-Terra C-18 column. A binary gradient system with a flow rate of 1.0 mL/min was dissolved using a 0.1% elution solvent, 5% acetonitrile, 0.1% acetonitrile, and 5% acetonitrile.
2.7 In vivo excretion
The mice were anesthetized by exposure to 2 % isoflurane with oxygen. Small animal PET images were taken in the mice with Sc-44, Sc-44-DOTA and Sc-44-DOTA-[Nle]-cCCK at intervals of 1 hour and 17 hours after intravenous injection through the tail veins.
AR42J cells were plated on to a 12-well culture plate with 1 ml of RPMI-1640 growth medium at a density of 1×105 and cultured at 37 °C and 5 % CO2 for 24 hours in a humidified atmosphere of 95% air. The growth medium was replaced by FBS containing a phenol red pre-experimental medium and 10 μl of DMSO. The treatment lasted 24 hours. At the end of the culture, the cytotoxicity of the compound was evaluated by a dye absorption test using crystal violet as described above . Optical density measurements were obtained at 540 nm using a microplate reader. The average absorbance value of the control device was considered as 100% cell viability.
2.9 Statistical analysis
All values are expressed as the mean ± standard deviation (SD). Student’s t-test was used to evaluate differences between groups and the two-sided Spearman’s correlation coefficient was used to determine the relationship between variables. A probability of less than 0.05 was considered to be statistically significant.