Cyclone plus system

To evaluate the time course of radiolabeling, 45 µL of Fe₃O₄@Al(OH)₃ NPs containing 60 µg of Fe were incubated with 5–30 MBq [¹⁸F]NaF (5–30 µL) while shaking. Two, five, and 10 min after labeling, 2 µL samples were taken and blotted on instant thin layer chromatography (iTLC) papers impregnated with silica gel (iTLC-SG papers; Varian, Diegem, Belgium). The papers were developed in an elution chamber using NaCl 0.9% as the mobile phase. The read-out was performed using a 2480 Wizard² Automatic Gamma Counter (20 s protocol; PerkinElmer, Waltham, MA, USA) after splitting the papers into two equal halves representing the unbound and bound radiotracer. In addition, autoradiography was performed using phosphor screens (Perkin Elmer) in standard film cassettes. The screens were removed from the cassettes after a five-minute exposure and were scanned immediately at 300 dpi resolution using a Cyclone Plus System (Perkin Elmer). Images were analyzed using the manufactures’ Optiquant software (version 5; Perkin Elmer).
For further in vitro and in vivo experiment, NPs are labeled with [¹⁸F]NaF for ten minutes in Milli-Q water. Afterward, they are centrifuged for 20 min at 4000 rpm and resuspended in the media of choice for further application.

Wild-type mice were anesthetized (2.5% isoflurane in O₂ at 1 L/min flow rate) and intravenously injected with [¹⁸F]AlF-NOTA-DV1-k-(DV3) and [⁶⁸Ga]Ga-DOTA-DV1-k-(DV3) (3.5–4 MBq). After 75 min, mice were sacrificed by decapitation. Organs of interest were quickly excised and snap frozen in 2-methylbutane (−40 °C). Next, 20 μm sections of liver, kidney and spleen were obtained using a cryotome (Shandon cryotome FSE; Thermo Fisher Scientific, Waltham, MA, USA) and these were mounted on adhesive microscope slides (Superfrost Plus; Thermo Fisher Scientific, Thermo Fisher Scientific, Waltham, MA, USA) after which they were exposed to a phosphor storage screen (super-resolution screen; Perkin Elmer, Waltham, MA, USA) overnight. Screens were read in a Cyclone Plus system (Perkin Elmer, Waltham, MA, USA), and images were analyzed using OptiQuant software (Perkin Elmer, Waltham, MA, USA). The remaining excised tissue was further sectioned in 20 μm slices and stored at −20 °C.

Optimization of radiolabeling conditions: [¹⁶¹Tb]TbCl₃ (0.2 MBq, 10 μL, 50 mM HCl) was added to 90 μL of a solution with different quantities of the ligand (DTPA, DOTA, DOTA-GA or NETA, 0.1-1.0 nmol) in sodium acetate buffer (0.1M, pH 4.7, chelex treated) and reacted in a glass vial for 60 min at 25 or 40°C (n = 3). The radiochemical yield of each reaction mixture was determined by instant thin-layer liquid chromatography (iTLC-SG, Varian, Diegem, Belgium). iTLC-SG papers were developed in an elution chamber using acetonitrile/water (75/25). The distribution of activity on the iTLC chromatograms was quantified using phosphor storage autoradiography [super-resolution screen, Perkin Elmer, Waltham, USA processed in a Cyclone Plus system (Perkin Elmer) and analyzed using Optiquant software (Perkin Elmer)].
Radiolabeling HSA-constructs: Purified HSA-constructs were labeled using 10 μM of the HSA-conjugate (90 μL) with [¹⁶¹Tb]TbCl₃ (0.2 MBq, 10 μL, 50 mM HCl) at 40°C for 60 min (n = 3). Radiochemical yields were determined by iTLC-SG, eluted with sodium citrate buffer (0.1 M, pH 5.8). Radiolabeled HSA-constructs were additionally analyzed by radio-SEC using the method described above.