Chemicals
Enriched 46CaCO3 (83.09% 40Ca, 1.19% 42Ca, 0.36% 43Ca, 8.55% 44Ca, 5.00 ± 0.50% 46Ca, 1.81% 48Ca, Trace Sciences International, USA) was used as target material for thermal neutron irradiation. Enriched 47TiO2 (0.41% 46Ti, 95.7 ± 0.3% 47Ti, 3.61% 48Ti, 0.15% 49Ti, 0.13% 50Ti, Isoflex, USA) was reduced to 47Ti metal and used as target material for fast neutron irradiation. Prior to irradiation, a precursory scan for trace metals by ICP-OES (Perkin Elmer Optima 3000) was performed.
The chemical separation of Sc from Ca was performed on a N,N,N’,N’-tetra-n-octyldiglycolamide, non-branched resin (DGA, particle size 50–100 μm, TrisKem International, France). SCX cation exchange cartridges (100 mg Bond Elut SCX, particle size 40 μm, Agilent Technologies Inc., USA) or DGA extraction chromatographic resin were used for the preconcentration of Sc. Chemical separations were performed with MilliQ water, hydrochloric acid (HCl, 30% Suprapur, Merck KGaA, Germany) and sodium chloride (NaCl, Trace Select, ≥99.999%, Fluka Analytical, Germany). For the 47TiO2 reduction process, calcium hydride (CaH2, metals basis, Mg <1%, Alfa Aesar, Germany), argon (Ar, 99.9999%, Linde, Germany) and acetic acid (CH3COOH, 100% Suprapur, Merck KGaA, Germany) were used. Nitric acid (HNO3, 65% Suprapur, Merck KGaA, Germany) was required for the preparation of the 46Ca targets. DOTANOC acetate was obtained from ABX GmbH, advanced biochemical compounds, Germany.
Production of 47Sc from enriched 47Ti
The reduction of 47TiO2 was performed at Helmholtz Center for Heavy Ion Research (GSI) in Darmstadt as described elsewhere (Lommel et al. 2014). Briefly, the enriched 47TiO2 was combined with 40% surplus of calcium hydride and the reduction process was performed under constant argon flow at 900 °C for 1 h. Dilute acetic acid was used for the isolation of the reduced 47Ti metal from the co-produced calcium oxide.
To prepare the targets, 0.6–19.9 mg reduced 47Ti powder was placed in a quartz glass ampoule (Suprasil, Heraeus, Germany) and sealed. The targets were irradiated with neutrons at the spallation-induced neutron source, SINQ, at Paul Scherrer Institut (PSI) at a fast neutron flux (>1 MeV) of 3.3–3.5 × 1011 n cm−2 s−1 for 1.5–18.9 days and in the BR2 reactor at SCK.CEN, Mol, Belgium in a reflector channel at a fast neutron flux (>1 MeV) of 5.7 × 1013 n cm−2 s−1 for 7 days. 47Sc was formed via the 47Ti(n,p)47Sc nuclear reaction with fast neutrons.
Production of 47Sc from enriched 46Ca
To prepare the targets, 65–91 mg enriched 46CaCO3 powder was dissolved in concentrated nitric acid and evaporated to complete dryness at 60–70 °C. The 46Ca(NO3)2 residue was taken up in dilute nitric acid (~1 M HNO3) and an aliquot of the aqueous solution (0.14–0.35 mg 46Ca) transferred into a quartz glass ampoule, evaporated to dryness and sealed.
47Sc was produced by the irradiation of the described 46Ca targets with thermal neutrons at the high flux reactor of Institut Laue-Langevin (ILL) in Grenoble, France at a thermal neutron flux of 1.0–1.4 × 1015 n cm−2 s−1, for 4 to 11 days, and at the BR2 reactor at SCK.CEN, Mol, Belgium at a thermal neutron flux of 3.2 × 1014 n cm−2 s−1 for 7 days, respectively. 47Sc was generated by the decay of the formed 47Ca (T1/2 = 4.54 d) occurring during the irradiation, but also after removal of the ampoule from the reactor.
Separation of 47Sc from 46Ca and 47Ca
The irradiated 46Ca ampoules were delivered to PSI several days post-irradiation (2.6–12.4 d) and the 47Sc separation was performed immediately, similarly to previously reported (Müller et al. 2014a). Each ampoule was transferred into a hot cell and the glass surface was cleaned twice with ~20 mL 1.0 M HCl and rinsed twice with ~20 mL MilliQ water. The crushing of the quartz glass ampoule was performed within a plastic target tube in a separate hot cell. Subsequently, the target tube containing the crushed ampoule was attached to the separation panel with the aid of manipulators. The design of the separation panel, including the adaptation of its operation inside the hot cell, was a crucial part of the method development (Fig. 2). The 46Ca(NO3)2 (~10–25 mg) from the ampoule was dissolved in 4 mL 3.0 M HCl and transferred from the target tube to the reaction vessel. A system of syringes, peristaltic pumps and three-way valves (see schematic of the panel in Fig. 2) was used to transfer the reagents from outside into the hot cell. To ensure complete dissolution of the target material, the solution was pumped from the target tube to the reaction vial and back several times. The solution was loaded on a pre-conditioned DGA column (1 mL cartridge filled with 50–70 mg of DGA resin). A second rinse cycle of the crushed glass ampoule with 2.5 mL 3.0 M HCl ensured collection of final traces of the 47Sc activity, which were subsequently sorbed onto the DGA resin column. Radioactivity detection probes were attached in the vicinity of the target tube and the DGA column to follow the transfer of the 47Sc radioactivity. Further application of 2 mL 3.0 M HCl removed the stable 46Ca and radioactive 47Ca from the DGA resin. The entire Ca-containing effluent was collected in a separate vessel and kept for consecutive separation of further in-growing 47Sc from the decaying 47Ca. The sorbed 47Sc was eluted from the resin column with 4 mL 0.1 M HCl and sorbed on a second column containing SCX cation exchange resin (Method A). Alternately, the 47Sc-containing eluate was collected, acidified to yield a 3.0 M HCl solution and sorbed on a second, smaller DGA resin column (1 mL cartridge filled with 20–25 mg DGA resin) at a slow flow rate of ~0.3 mL/min (Method B), as described by Domnanich et al. (Domnanich et al. 2016). The elution of 47Sc from the second column was performed with 700 μL 4.8 M NaCl/0.1 M HCl (for Method A) and with 1.7 mL 0.05 M HCl (for Method B) via a separate valve. In order to collect 47Sc in a small volume, the 0.05 M HCl (Method B) was fractionized into three Eppendorf vials; the first contained ~700 μL and the other two ~500 μL each. Fractionized collection was not necessary for Method A, as the highest proportion of the 47Sc radioactivity was trapped in a low quantity of eluate.
The renewed generation of 47Sc, by the decay of radioactive 47Ca in the Ca-containing fraction (47Ca and stable 46Ca), enabled subsequent separations after a minimum in-growth time of 3 days.
Radionuclidic purity
To identify the nuclide inventory of the samples, γ-ray spectrometry with an N-type high-purity germanium (HPGe) coaxial detector (EURISYS MESURES, France) and the Ortec InterWinner 7.1 software were employed. The aliquot of 47Sc eluate was in the range of 3–15 MBq, while the entire neutron irradiated glass ampoules containing the 47Ti were used for the measurements. The counting time was determined by ensuring the measurement error was <4%. To determine small activities of long-lived radionuclidic impurities, γ-spectrometry measurements of the same samples were performed with an extensive counting time several days post-irradiation.
Radiolabeling for quality control of the produced 47Sc
After quantitative determination of the 47Sc activity in the eluate with a dose calibrator (ISOMED 2010, Nuclear-Medizintechnik Dresden, GmbH, Germany), the required activity for radiolabeling in 0.05 M HCl was withdrawn from the product vial and 0.5 M sodium acetate solution (pH 8) was added to the 47Sc eluate to obtain a pH value of ~4.5. DOTANOC (0.7 mM solution in MilliQ water) was added to the 47Sc solution (~50 MBq) to obtain a specific activity of 10–25 MBq/nmol (2–5 nmol DOTANOC). The reaction mixture was incubated at 95 °C for 15 min. The preparation of 177Lu-DOTANOC (25 MBq/nmol) was carried out under standard labeling conditions (pH 4.5, 95 °C) using no carrier-added 177Lu (purchased from Isotope Technologies Garching GmbH, Germany) (Müller et al. 2013a).
High-performance liquid chromatography (HPLC, Merck Hitachi, LaChrom) with a C-18 reversed-phase column (XterraTM MS, C18, 5 μm, 150 × 4.6 mm; Waters) was used for determination of the radiolabeled fraction of DOTANOC. The detection was performed with a UV (LaChrom L-7400) and radiodetector (Berthold, HPLC Radioactivity Monitor, LB 506B). The mobile phase consisted of MilliQ water containing 0.1% trifluoracetic acid (A) and acetonitrile (B) with a gradient of 95% A and 5% B to 20% A and 80% B, over a period of 15 min, at a flow rate of 1.0 mL/min.
In vitro stability of 47Sc- and 177Lu-labeled DOTANOC
The in vitro stability of 47Sc- and 177Lu-labeled DOTANOC (radiochemical purities >95%) was investigated in phosphate buffered saline (PBS, pH 7.4). An activity of 50 MBq of 47Sc- or 177Lu-DOTANOC was diluted with PBS (pH 7.4) to a total volume of 500 μL and incubated at room temperature for 3 days. Once every 24 h an aliquot was withdrawn to determine the integrity of the labeled compound by means of HPLC.
SPECT/CT imaging with 47Sc- and 177Lu-DOTANOC
In vivo experiments were approved by the local veterinarian department and conducted in accordance with the Swiss law of animal protection. Female, athymic nude mice (CD-1 nude) at the age of 5–6 weeks were obtained from Charles River Laboratories, Sulzfeld, Germany. AR42J cells (rat exocrine pancreatic tumor cells, European Collection of Cell Cultures ECACC, Salisbury, U.K.) were suspended in PBS (5 × 106 cells in 100 μL) and subcutaneously inoculated on each shoulder. SPECT/CT experiments were performed about 2 weeks after tumor cell inoculation, when the tumor reached a size of about 400 mm3.
Imaging studies were performed using a small-animal SPECT camera (NanoSPECT/CT™, Mediso Medical Imaging Systems, Budapest, Hungary) as previously reported (Müller et al. 2014b). The energy peaks for the camera were set at 159.4 keV (± 10%) for the scans with 47Sc and 56.1 keV (± 10%), 112.9 keV (± 10%) and 208.4 keV (± 10%) for the scans with 177Lu. SPECT/CT scans were followed by CT scans. The images were acquired using Nucline Software (version 1.02, Bioscan Inc., Poway, California, US). The reconstruction was performed iteratively with HiSPECT software (version 1.4.3049, Scivis GmbH, Göttingen, Germany). SPECT and CT data were automatically co-registered and the fused datasets were analyzed with the VivoQuant post-processing software (version 2.50, inviCRO Imaging Services and Software, Boston, USA).
The mice were injected intravenously with 47Sc-DOTANOC (12 MBq, 1.2 nmol, 100 μL) and 177Lu-DOTANOC (40 MBq, 1.2 nmol, 100 μL), respectively. The in vivo SPECT/CT scans of 35 min duration were acquired 3 h after injection of 47Sc-DOTANOC. During the scans, the mice were anesthetized by inhalation of a mixture of isoflurane and oxygen. Post-mortem scans of 1.3–3.5 h were performed 6 h after injection of 47Sc- and 177Lu-DOTANOC. The SPECT acquisitions were performed in such a manner to obtain the same total number of counts for each scan.