Chemicals and reagents
NaOH micropills and acetic acid were purchased from POCH S.A. Gliwice. Ammonia (ammonium hydroxide solution 25 %), citric acid, and ammonium carbonate were purchased from Sigma Aldrich. N,N,N′,N′-tetra-n-octyldiglycolamide (DGA) 50–100 mesh and UTEVA 100–200 mesh resins were purchased from Eichrom, USA; Chelex 100 resin (Na+ form, mesh size 100–200) was purchased from Bio-Rad, USA; and DOWEX 50 × 8 resin (hydrogen form, 200–400 mesh) was purchased from Fluka Analytical, Germany. [DOTA,Tyr3] octreotate (DOTATATE) 95 % purity (HPLC) was purchased from piChem (Graz, Austria). All chemicals were of analytical grade and were used without further purification.
Natural CaCO3 of chemical purity >99.999 purchased from Sigma Aldrich and enriched [40Ca]CaCO3 (99.99 %) purchased from Isoflex (USA) were used as target materials. The isotopic composition of enriched 40Ca was 99.99 % of 40Ca and 0.01% of 44Ca, while the amounts of other calcium isotopes were below 0.01 %.
Irradiation of natCaCO3 and [40Ca]CaCO3 targets
Irradiations of natural targets were performed using the Scanditronix MC 40 cyclotron at the European Commission’s Joint Research Centre (Ispra, Italy). Irradiations of enriched [40Ca]CaCO3 targets were performed using the Warsaw Heavy Ion Cyclotron operating at the Heavy Ion Laboratory of the University of Warsaw. The Ispra cyclotron is capable of accelerating positive ions such as protons, deuterons, and alphas to variable energies. The Warsaw machine accelerates heavy ions from +He up to Ar with energies from 2 up to about 8 AMeV. For irradiation at the Ispra cyclotron, the target material was wrapped in an aluminum foil of a 25-μm thickness. The samples were irradiated in aluminum capsules with an inner diameter of 10 mm. Each target capsule was inserted in a holder that allowed direct water cooling from both the rear and the front sides. In the Warsaw cyclotron, targets in the form of pellets bundled in thin aluminum foils produced from CaCO3 powder using a hydraulic press were irradiated with an internal α-particle beam. Al energy degraders were used when alpha particle energies lower than maximal were necessary.
In order to optimize the yield of 43Sc production by the 40Ca(α,p)43Sc and natCa(α,n)43Ti→43Sc nuclear reactions, ~100-mg natCaCO3 samples (target thickness ~375 μm) were irradiated for 28–34 min by an alpha beam of 13–25 MeV on the target with an alpha current of 0.5 pμA at the Scanditronix MC 40 cyclotron.
Enriched [40Ca]CaCO3 targets of ~100 mg were irradiated for 30 min by an alpha beam of 20 MeV with an alpha current of 0.25 pμA (He+) at the Warsaw Heavy Ion Cyclotron.
Measurement of radioactivity
The absolute radioactivity of 43Sc and other obtained radionuclides was measured by γ-spectrometry using two high-purity germanium (HPGe) detectors. The detectors were energy and efficiency calibrated in different geometries using certified standard radioactive sources (ENEA Italy, DAMRI and CERCA France). The gamma-ray spectrum analysis software package Genie 2000 (CANBERA, USA) was used to collect the data. The γ-ray peak at 372.8 keV was chosen for 43Sc detection, and the peaks at 1157.00, 271.24, and 159.38 keV were chosen to detect 44Sc, 44mSc, and 47Sc, respectively. Three peaks at 983.52, 1037.52, 1312.10 keV were used to quantify yields of 48Sc [21]. The uncertainty of all the determined activities was below 1 %.
Separation of 43Sc from the target
In order to find the optimal method for the separation of 43Sc from the irradiated calcium targets, three procedures were tested:
In the first method, described by Valdovinos et al. [22], the irradiated natCaCO3 target was dissolved in 1 ml of 9 M HCl solution. The dissolved target solution was passed through a column containing 50 mg of UTEVA resin, and after adsorption of 43Sc, the column was washed with 5 ml of 9 M HCl. The scandium radionuclides were eluted with a 400-μl portion of H2O.
The second method, reported in the paper by Mueller et al. [10], consists of dissolving the CaCO3 targets in 3 M HCl and adsorption of scandium radionuclides in a column filled with 70 mg of DGA. The adsorbed 43Sc was eluted from the DGA resin with HCl (0.1 M, 2–3 ml). Afterwards, the acidic 43Sc solution was loaded on a second column filled with 100 mg of cation exchange resin DOWEX 50 (hydrogen form, 200–400 mesh). Finally, 43Sc was eluted using 1 M ammonium acetate adjusted to pH = 4 using HCl solution.
The third method, developed by our group [9], consisted of dissolution of the target in 1 M HCl and adsorption of 43Sc on chelating ion exchange resin Chelex 100 of bed size 0.8 × 4.0 cm and conditioned with 5 ml of 1 M HCl. After adsorption of 43Sc and Ca2+, the column was washed with 30 ml of 0.01 M HCl in order to remove Ca2+. The scandium radionuclides were then eluted with 1 M HCl in 0.5-ml fractions.
Radiolabeling and stability studies of DOTATATE conjugate
DOTATATE, octreotate-somatostatin analog conjugated to DOTA chelator, was labeled with the obtained 43Sc using 10, 15, and 25 nmol of the peptide. The most active fraction of 43Sc solution was combined with 0.2 ml of 0.2 M sodium acetate buffer (pH = 6) containing 14, 21, or 36 μl of the peptide (0.7 nmol μl−1) in the buffer. The solution was next heated for 25 min at 95 °C in a water bath. Product formation and reaction yields were estimated by instant thin-layer chromatography (ITLC) using Silica gel 60 TLC plates (Merck). A 0.1 M citric buffer of pH = 5.4 was used as the eluent. Of the solution, 10 μl was dropped on the ITLC strip. Free 43Sc moved with the front boundary of the solution whereas the labeled bioconjugate remained at the starting point. The labeling yield defined as the percentage of 43Sc radioactivity complexed by DOTATATE to the starting activity was calculated as the ratio of the activity of the strip application part to the whole strip activity.
The stability of the labeled DOTATATE in human serum was assessed by adding 20 μl of the radioconjugate solution to 500 μl of the human serum. The mixture was incubated at 37 °C, and the stability was measured by taking aliquots of the human serum solutions at different times and measuring the liberated scandium radionuclide by ITLC analysis.