General
FSH4 peptide (YTRDLVYKDPARPKIQKTCTFNDRGGG) and FSH1 peptide were obtained from Apeptide Co., Ltd. (Shanghai, China). Malemide-NOTA (denoted as MAL-NOTA) was purchased from CheMatech (Dijon, France). Aprotinin was bought from Sigma-Aldrich. [68Ga]GaCl3 was obtained by eluting a 68Ge/68Ga-generator (ITG, Germany) with 0.05 M hydrochloric acid. All reagents were analytical grade and employed without further purification. HPLC systems for analysis and purification peptides were the same as the literature report.[14, 15, 24] The animal experiments were performed according to the national regulations and approved by the animal welfare committee.
NOTA Conjugation of Peptides
FSH4 (3mg, 0.96μmol) was added to a vial and followed by 3ml Malemide-NOTA (0.5mg, 1,1μmol) in 2M ammonium acetate solutions. Then the mixture was stirred at 40 °C overnight. After purification with HPLC, the desired product was collected and lyophilized as a white powder. The mass spectrum was determined using a high resolution LC-MS system(Waters, Milford, USA).
Preparation of 68Ga-NOTA-MAL-FSH4
Labeling the peptide with 68Ga was carried out in an Eppendorf vial containing NOTA-MAL-FSH4 (20μg, 6.7 nmol). Fresh [68Ga] GaCl3 (185 MBq) eluate was added to the vial followed by 0.25M sodium acetate solutions. After incubating at 100 °C for 10 min, the complex was diluted with deionized water and then loaded into a Varian BOND ELUT C18 column. The labeled peptide was obtained after eluting the column with 200μL 10 mM HCl in ethanol. The product was reconstituted in saline and sterilized using a 0.22 μm Millipore filter. Radiochemical purity was analyzed with HPLC.
Cell lines and animal models
PC-3 human prostate cancer cells were purchased from Cell Bank of Shanghai Institutes for Biological Sciences. The cells were cultured in DMEM (Gibco, USA) and supplemented with 10% (v/v) fetal bovine serum and kept in a humidified atmosphere containing 5% CO2 at 37℃.
Male Balb/c nude mice (4 weeks old) were obtained from CAVENS Laboratory animal co.ltd, China. A suspension of 5×106 PC-3 cells in sterilized saline was subcutaneously injected into the right front flank of the mice. When the tumors developed proper sizes (100–300 mm3), the following animal experiments were performed.
Cell Binding Assay
PC-3 human prostate cancer cells (2×105 cells) were seeded in 6 wells plates and cultured at 37℃ overnight. After washing with binding buffer (RPMI, 0.5 % bovine serum albumin), 68Ga-NOTA-MAL-FSH4 ( 37 KBq) and FSH1 peptide ranged from 0 to 5,000 nM were added to each well. The medium was removed after incubation for 2 hours at 37 °C. After washing 3 times with PBS, the cells were lysed using 1M NaOH and the radioactivity was determined in a γ-counter (PerkinElmer). Inhibitory concentration of 50 %(IC50) values were calculated by GraphPadPrism software. Experiment was performed in triplicate.
In vitro metabolism analyses
Mice bearing PC-3 tumors were sacrificed and the main organs (blood, liver, kidney and tumor) were collected. After centrifugation at 4℃, plasma was obtained from the blood. Other organs were homogenized with ice PBS at 4℃. Each plasma or tissue homogenate was added 37KBq 68Ga-NOTA-MAL-FSH4 with or without 100μg aprotinin and incubated 30 min at 37℃. Plasma and tissue homogenates were denatured with an equivalent volume of acetonitrile in centrifuge tubes. After vortexing, the mixture was centrifuged and the supernatant was separated and analyzed by HPLC.
In vivo MicroPET imaging
Mice (n=4 per group) were injected with 200 μL 3.7 MBq 68Ga-NOTA-MAL-FSH4 in saline via a tail vein. After anesthetized with isoflurane, the mice were performed PET imaging using a microPET scanner (Inveon, Siemens). At 30min, 60 min and 120min postinjection, static PET images were acquired. To investigate the effects of enzyme inhibition by aprotinin, 100 μL 3.7 MBq 68Ga-NOTA-MAL-FSH4 together with 100μL aprotinin (100μg, 200μg or 400μg respectively) in saline were injected into mice via a tail vein. Static PET images were acquired for 10 min at 30min, 60 min and 120min after injection. The quantification analysis of PET images was carried out according to the previous method.[14, 15, 24]
Ex vivo biodistribution experiments
Mice bearing PC-3 tumors were injected with about 740KBq radiolabeled peptides in the absence or presence of 400μg aprotinin and sacrificed at selected time points. Blood, tumor and major organs were collected and weighed. The radioactivity was measured by a γ-counter. Data were determined by the percentage injected dose per gram of tissue (%ID/g). Blocking studies were performed through coadministration of excessive unlabeled FSH1 with the tracer.
Statistical analysis.
One-way analysis of variance (ANOVA) and Student’st test were used for statistical analysis of data. P values of < 0.05 were considered to be statistically significant.