The chemicals used were all analytical reagent grades and used without further purification unless stated. Acetonitrile (ACN) and dimethylformamide (DMF) were kept over molecular sieves. High Pressure Liquid Chromatography (HPLC) analysis was carried out on Econosil C-18 reversed phase columns (semipreparative, 250 mm x 10 mm or analytical, 250 mm x 4.6 mm). The solvent system used for the semipreparative was non-linear gradient (eluant A, H2O with 0.1% trifluoroacetic acid (TFA); eluant B, ACN/H2O, 3/1 v/v with 0.1% TFA; gradient, 0 to 90% B, 90 to 90% B and 90 to 10% B over 10 min each at flow rate of 1.5 mL/min) and for the analytical was (eluant A, ACN with 0.1% TFA; eluant B, H2O with 0.1% TFA; gradient, 0 to 50% B, over 0–15 min and 50 to 0% B over 15–20 min at flow rate of 1.5 mL/min). A Jasco chromatographic system equipped with a variable wavelength ultraviolet monitor and in tandem with a Canberra flow through radioactivity detector was used. Ultraviolet absorption was monitored at 220 nm. Chromatograms were acquired and analyzed using BORWIN software. Mass spectroscopy was run on Quattra electrospray mass spectrometer (ES-MS).
2.1. MUC1and MUC1-FA hybrid peptide conjugates
The MUC1 peptide analog was prepared utilizing the method reported previously [31]. Briefly, by solid-phase peptide synthesis (on a CS Bio peptide synthesizer, CA, USA) following standard Fmoc (9-fluorenylmethoxycarbonyl) chemistry, using Rink amide methylbenzhydrylamine (MBHA) resin on a 0.2 mmol scale. After incorporating all the desired amino acids, the N-terminal Fmoc-protecting group was removed and the peptide was cleaved from the resin followed by the removal the other side-chain protecting groups using mixture of TFA/H2O/dithiothreitol (DTT) 95:2.5:2.5 for 2 h at room temperature. The resin was removed by filtration, and the crude peptides obtained by precipitation with cold diethyl ether (ether) followed by HPLC purification. For the synthesis of MUC1-FA hybrid peptide, the free epsilon (ɛ) amino group at terminal Lys residue on MUC1 peptide was coupled with FA via the activated gamma (γ) carboxyl moiety. The N-succinimidyl folate ester (folate-NHS, 10 µmol) dissolved in dimethylsulfoxide (DMSO, 50 µL) and followed by the addition of each peptide (10 µmol) and TEA (10 µmol). Reaction mixture stirred while shielded from light for 30 min at 50o C. The MUC1-FA hybrid peptide was precipitated by addition of ACN (2 mL), centrifuged and then washed several times with ACN before drying. The identity and purity of the MUC1 peptide analog was characterized by mass spectrometry and HPLC.
2.2. Fluorinated MUC1- and MUC1-FA-SFB hybrid peptide reference conjugates
The reference fluorinated MUC1 and MUC1-FA peptide conjugates were prepared separately by coupling the precursors 4-fluorobenzoic acid to the non-receptor binding region through an amide linkage utilizing the methods reported previously [31, 32] (Scheme 1). Briefly, MUC1 and MUC1-FA hybrid peptides (5 µmol each) were added to N-succinimidyl-4-fluorobenzoate (SFB, 8.5 µmol) in DMF (100 µL) and enough amount of triethylamine (TEA, 2 µL) to attain a pH 9. The reaction mixtures were heated for 20 min at 90o C. This was followed by dilution with H2O (1 mL), loading onto Sep-Pak C-18 cartridge, washing with H2O (5 mL) and the peptide conjugate eluted with ethanol (EtOH, 1 mL). After solvents evaporation to dryness, white and slightly yellowish powders were separated and dried under vacuum to produce the reference MUC1-SFB or MUC1-FA-SFB hybrid peptide conjugates, respectively. The structures and purities of the fluorinated peptide analogs were characterized by mass spectrometry and HPLC.
2.3. MUC1 and MUC1-FA 4-(N,N,N-trimethylammonium)benzoate.triflate hybrid peptide precursors
The MUC1 and MUC1-FA hybrid peptide conjugates (3 mg, 3.5 µmol) were dissolved separately in DMF (100 µL) followed by the addition of TEA (1 µL, 6 µmol). N-succinimidyl 4-(N,N,N-trimethylammonium)benzoate.triflate (1 mg, 3.6 µmol) was then added and mixtures stirred at 80o C for 15 min (Scheme 2). The MUC1- and MUC1-FA-triflate precursors were precipitated by addition of ACN (1 mL), centrifuged and then washed several times with ACN before drying under vacuum to yield white and slightly yellowish powders in 59% and 66%, respectively.
2.4. Radiosynthesis of MUC1-[18F]- and MUC1-FA-[18F]SFB hybrid peptide conjugates
Aqueous [18F]-fluoride was produced by the 18O(p,n)18F reaction. The fluoride activity (2–8 mCi, 74–296 MBq) was trapped in Kryptofix 2.2.2 (5 mg) and potassium carbonate (1 mg) in ACN/H2O solution (950 µL/50 µL), dried by azeotropic distillation with aliquots of ACN. The solid residue was resolubilized in DMF (0.2 mL) and reacted in two different sealed vials containing the precursor Mucin 4-(N,N,N-trimethylammonium)benzoate.triflate peptide (50 µg, 50 nmol) and MUC1-FA 4-(N,N,N-trimethylammonium)benzoate.triflate hybrid peptide (50 µg, 35 nmol). The reaction mixtures were heated in capped 2 mL reaction-vials at 90o C for 5 min, followed by the addition of H2O (1 mL) then passed through Sep-Pak C18 cartridge and washed with H2O (5 mL) to remove hydrophilic impurities (Scheme 3,4). Sep-Pak C18 cartridge was then dried with steady stream of nitrogen, MUC1-[18F]SFB and MUC1-FA-[18F]SFB hybrid peptide conjugates were eluted with EtOH (1 mL). EtOH solutions were dried and residues were then re-solubilized in saline (NaCl 0.9%, 1 mL each) before passing through 0.22 µm pore membrane filter for further studies.
2.5. Partition Coefficient
100 µL of MUC1-[18F]SFB and MUC1-FA-[18F]SFB hybrid peptide conjugates were added into test tubes containing 1 mL of each n-octanol and buffered H2O (pH = 7.3). The tubes were shaken for 1 min. After partial separation of the phases by gravity, 0.7 mL of each phase was transferred to separate tubes and centrifuged at 5000 rpm for 5 min. Duplicate 0.2 mL aliquots of each phase was taken for radioactivity measurement and the partition coefficient was determined by the function: Partition coefficient = Log10 (counts in n-octanol layer/counts in aqueous layer).
2.6. Stability in Plasma
For stability in plasma, the purified MUC1-[18F]SFB and MUC1-FA-[18F]SFB hybrid peptide conjugates (100 µL, 20 µCi each) were incubated with human plasma (500 µL) in duplicate at 37o C for 2 h. This was followed by precipitation using a mixture of ACN/EtOH (400 µL, 1:1 v/v) and centrifugation at 5000 rpm for 5 min. The supernatant layer was then analyzed by HPLC under the conditions described above.
2.7. In Vitro Cell Binding
The cell-binding activity of the MUC1-[18F]SFB and MUC1-FA-[18F]SFB hybrid peptide conjugates were measured on human MCF7 breast cancer cell line (ATCC, Rockville, MD). MCF7 cell line was grown in RPMI-1640 culture media with 10% fetal bovine serum (FBS) in tissue culture flasks. 24 h prior conducting the cell-binding assay, media was replaced with RPMI-1640 without further addition of FBS. Confluent cultures were harvested by trypsinization, and 6 × 106 cells were suspended in 1.8 mL of sterile saline for binding assay. Approximately 300,000 cells (in 0.3 mL of sterile saline) were incubated with various amounts of the purified MUC1-[18F]SFB and MUC1-FA-[18F]SFB hybrid peptide conjugates ranging from 0.3–18 nM in duplicate for 60 min at room temperature. Incubation was terminated by dilution with cold saline (0.3 mL) and cells were pelleted by centrifugation. The cell-pellets were then washed with cold saline to remove unbound radioactivity and centrifuged to collect supernatants. Radioactivity in the cell-pellets (total bound) and washings (unbound) were measured in a γ-well counter. Non-specific binding, was determined in the presence of approximately 100-fold excess of unlabeled MUC1 and MUC1-FA- hybrid peptide. Specific binding was calculated by subtracting the non-specific bound radioactivity from that of the total binding. The data were analyzed by a non-linear regression analysis program (Graph-Pad Software Inc., San Diego, CA, USA) using one-site binding equation. All binding data were corrected for non-specific binding and presented as the mean ± S.D.
2.8. In vivo Biodistribution
Approval for the animal protocol used in this study was obtained from the Institutional Animal Care and Use Committee. Animal biodistribution experiments were performed according to the international regulations governing the safe and humane use of laboratory animals in research [33]. The biodistribution was performed in normal female Balb/c mice (body mass 20–25 g) to ascertain the in vivo distribution profile of the MUC1-[18F]SFB and MUC1-FA-[18F]SFB hybrid peptide conjugates. Mice were injected via the lateral tail vein with 100 µL of the radiotracers formulated in saline. Each dose contained 20 µCi (740 kBq) of radioactivity. Animals were sacrificed at different time intervals and tissues of interest were dissected, weighed and assayed for radioactivity. The percentage of the injected dose per gram (% ID/g) was then calculated by counting all tissues in a γ-well counter using a stored sample of the injection solution as a standard to estimate the total dose injected per mouse.
2.9. In vivo tumor targeting
Human MCF7 xenografts SCID female mouse models were used for in vivo tumor targeting experiments. For the implantation of tumor xenografts, approximately 3 × 106 MCF7 cells in suspension of 100 µL sterile saline were injected subcutaneous into the right thigh of each mouse. Tumors were allowed to grow for 4–6 weeks by which tumors had reached weights of ~ 500 mg. Animals were injected with 20 µCi (740 kBq) of the radiotracers. For the blocking studies, each animal was intravenously injected with excess cold of MUC1 and MUC1-FA hybrid peptide (~ 100 µg) 30 min prior to the radiotracers injection. The animals (n = 4 per group) were sacrificed at 60 min post radiotracers injection (p.i.) and the % ID/g for the tumor and major organs was calculated as described above.
2.10 In vivo Nano PET/CT imaging
PET/CT scans were performed using a preclinical NanoPET/CT scanner (Mediso, Hungary) on MCF7 tumor-bearing SCID female mice (8 weeks old). MUC1-[18F]SFB and MUC1-FA-[18F]SFB hybrid peptide conjugates (7.4 MBq/100 µL) were injected into each mouse through tail vein and placed in the Nano PET/CT scanner with continuous O2 and 2% isoflurane supply. 20 min post tail vein injection of the radiotracers, the mouse was imaged for 30 min PET/CT acquisition time. Static scan was acquired at 60 min post- injection. CT scan was performed using the following parameters: X-ray voltage = 50 kVp, Exposure time = 300 ms. A total projection of 288 projects over 360° of rotation were acquired and reconstructed using a cosine filter. This was followed by a PET data acquisition with following parameters: 5-ns coincidence window and 400–600 keV energy window in 1–5 coincidence mode. Crystal efficiency correction was also applied, with a ring difference of 8, and the images were reconstructed by a three-dimensional ordered-subsets; exception maximum algorithm (subsets, 4; iterations, 6). Pixel size was 0.3 mm. The acquired data in these studies were analyzed by InterVeiw FUSION software developed by Mediso.
2.11. Statistical analysis
Data are expressed as mean ± S.D. where appropriate. For data comparisons, a Student’s t test was performed of the mean values using Graph-Pad Software (Graph-Pad Software Inc., San Diego, CA, USA). A probability value of P < 0.05 was considered statistically significant.