Synthesis of Radioactive Metal-FHs Using HIR Conditions

The HIR procedure was described in previous studies.²⁹ Some modifications such as heating vortex (HV) mixing (Figure S1), temperature elevation, and shortened reaction time were made to the HIR technique to further optimize radiolabeling of FH with the therapeutic (⁹YY³⁺ and [¹⁷⁷Lu]Lu³⁺) and relatively short half-life ([⁶⁴Cu]Cu²⁺) isotopes. Similar SEC purification steps to the nonradioactive metal-FH were utilized for the [¹⁷⁷Lu]Lu-FH, [⁹⁰Y]Y-FH and [⁶⁴Cu]Cu-FH syntheses. All of the radiochemical analysis results were decay corrected. In addition, the radiochemical yield (RCY) and purity (RCP) of radiolabeled FH (radio-FH) products were obtained by the division of the activity of the radio-FH by the amount of activity used at the beginning.²⁹

After dilution of the original [⁹⁰Y]YCl₃ in HCl (0.1 M), two identical samples were prepared. For each sample, the reaction mixture was prepared by adding [⁹⁰Y]YCl₃ (38 µL, A₀ 30 MBq, m_Y 0.0166 nmoles, N_Y,atoms 10¹³) and CTW (50 µL) into a 0.3 mL glass vial. The pH of the reaction mixture was adjusted to 8–9 by adding Na₂CO₃ (40 µL, 1 M in CTW) gradually. FH (33.3 µL, 1 mg Fe) was added to the reaction mixture and the total volume was brought up to 250 µL by adding CTW (88.7 µL). The final pH of the reaction mixture was reassured to be 8–9 (Supplementary Figure S1a and b). The glass vial was placed in a heating vortex silicon oil bath (Supplementary Figure S1c and d) and the reaction mixture was heated for 2 hrs at 120–130°C under vortexing. The reaction mixture was cooled down in an ice bath for 15 min under vortexing. DFO (5 µL, pH 7.5, 20 mM in CTW) was added to quench the reaction by reacting with any remaining free [⁹⁰Y]Y³⁺ and dissociating loosely bound [⁹⁰Y]Y³⁺ ions from FH. A PD-10 column was used for the SEC purification and the fraction collection was done as above for Groups 3 and 4 for nonradioactive metal-FH (Supplementary Figure S2a–d). The purified product was concentrated by centrifugation with a 50 kDa MC Amicon filter at 370 g (rcf) under room temperature (Supplementary Figure S3a–d). The final purified product was then collected in saline at 200 µL with approximately complete recovery (Supplementary Figure S3e). Finally, SEC analysis was performed for the purified product to calculate the RCP (Table 3) (procedure see below).

A Summary of Measured Decay Corrected RCY and RCP for Radiolabelled-FH Products*

Note: *Average of five replicates for [⁹⁰Y]Y-FH and three replicates for both [¹⁷⁷Lu]Lu-FH and [⁶⁴Cu]Cu-FH.

After dilution of the original [¹⁷⁷Lu]LuCl₃ in HCl (0.1 M), two identical samples were prepared. For each sample, firstly the reaction mixture was prepared by adding [¹⁷⁷Lu]LuCl₃ (20 µL, A₀ 15 MBq, m_Lu 0.0206 nmoles, N_Lu,atoms 1.2 10¹³) and CTW (50 µL) into a 0.3 mL glass vial (see Supplementary Figure S1e, f). The pH of the reaction mixture was adjusted to 8–9 by adding Na₂CO₃ (20 µL, 1 M in CTW) gradually. FH (33.3 µL, 1 mg Fe) was added to the reaction mixture and the total volume was brought up to 200 µL by adding CTW (96.7 µL). The final pH of the reaction mixture was reassured to be 8–9. The glass vial was placed in a heating vortex silicon oil bath and the reaction mixture was heated for 1 hr at 120–130°C under vortexing. The reaction mixture was cooled down in an ice bath for 15 mins under vortexing. DFO (5 µL, pH 7.5, 20 mM in CTW) was added to quench the reaction by reacting with any remaining free radioactive [¹⁷⁷Lu]Lu³⁺ and dissociating the loosely bound [¹⁷⁷Lu]Lu³⁺ ions from FH. A PD-10 column was used for the SEC purification and collected as above for Groups 3 and 4 for nonradioactive metal-FH. The purified product was concentrated by centrifugation with a 50 kDa MC Amicon filter for 370 g (rcf) under room temperature (Supplementary Figure S3a–d). The final purified product was then collected at 200 µL with approximately complete recovery (Supplementary Figure S3e). Finally, SEC analysis was performed for the purified product to calculate the RCP (Table 3) (procedure see below).

Three reaction mixtures were prepared for optimization of HIR conditions for higher Specific Activity (A_s) [⁶⁴Cu]Cu-FH syntheses. In general, the procedures were very similar to that for HIR syntheses of [¹⁷⁷Lu]Lu-FH and [⁹⁰Y]Y-FH except the variations indicated below including the amount of activities, temperature, reaction time, and stirring methods (Table 3).

Two samples with A₀ = 18 MBq and 1 mg Fe FH were prepared using similar procedures for [⁹⁰Y]Y-FH and [¹⁷⁷Lu]Lu-FH described above with heating temperature at 120°C for 2 hrs under magnetic stirring (Table 3).

Higher reaction rate was achieved by increasing the initial activity to A₀ = 22 MBq of [⁶⁴Cu]CuCl₂, reacting at an elevated temperature at 140°C, and shortening the reaction time to 1 hr under magnetic stirring (Table 3). The reaction rate for HRR-HIR and HIR was estimated using the cumulative activity equation⁴⁴ (1):

where , , and are the cumulative activity, the initial activity, the reaction time and the isotope half-life. The reaction rate calculations are exemplified in Supplement Materials.

The third set of [⁶⁴Cu]Cu-FH reaction mixture was prepared similar to HRR-HIR technique, except that the heating vortex (HV) technique (Figure S1) was used instead of magnetic stirrer for mixing the reaction mixture during the reaction (Table 3).

For both RCY and RCP analyses were performed using SEC and TLC methods. The SEC for RCY and RCP analyses recruited reported procedures.²⁹ For both [¹⁷⁷Lu]Lu-FH and [⁶⁴Cu]Cu-FH, the activity was counted by a gamma-counter, and decay-corrected elution curves were plotted for [¹⁷⁷Lu]Lu-FH (see Figure 3A, ​,BB and ​andE,E, ​,F)F) and [⁶⁴Cu]Cu-FH (see Figure 3C, ​,DD and ​andG,G, ​,H).H). For the analysis of [⁹⁰Y]Y-FH, a bremsstrahlung-based technique⁴⁵ had to be developed as [⁹⁰Y]Y is not a direct gamma emitter, and thus the conventional gamma-counter method cannot be used. The gamma-counter protocol was modified by setting the energy window to 2–2000 keV to detect the produced secondary bremsstrahlung radiation emitted by the beta particles. Then, the [⁹⁰Y]Y-FH SEC and TLC chromatogram were plotted (Figure 3A, ​,BB and ​andE,E, ​,F).F). In addition, to validate this technique the bremsstrahlung counts corresponding to 30 MBq [⁹⁰Y]YCl₃ (five replicates and the average value was used as the reference) and the activity of [⁹⁰Y]Y-FH product was measured and their ratio was compared against the ratio of the areas under the SEC and TLC chromatograms of [⁹⁰Y]Y-FH and [⁹⁰Y]Y-DFO. The ratio of area of [⁹⁰Y]Y-FH and [⁹⁰Y]Y-DFO under the curves also represents the RCY measured by a dose calibrator (ie, the ratio of [⁹⁰Y]Y-FH and [⁹⁰Y]YCl₃ activities) and thus the ratio of the bremsstrahlung counts corresponding to the [⁹⁰Y]Y-FH and [⁹⁰Y]YCl₃ activities should agree with the RCY measurements to validate the bremsstrahlung counts technique.

Characterization of crude reaction mixes and purified products for HIR-FH labeled with different radiocations. (A) The radiochemical yields (RCY) by SEC (PD-10 column) for reaction mixes yielding [⁹⁰Y]Y-FH or [¹⁷⁷Lu]Lu-FH are shown. Deferoxamine (DFO) is added to remove loosely bound radiocations from FH, and to ensure that radiocations are not present as oxides, but as low molecular weight, radio-DFO complexes. These complexes migrate at the included column volume in SEC or stay at the origin in TLC. See legend for 3e below. All values are decay corrected. Integrated areas under the peaks were used to obtain RCY or RCP. (B) Radiochemical purity (RCP) by SEC after reaction mixes from (A) was purified by SEC. (C) RCY by SEC for reactions yielding [⁶⁴Cu]Cu-FH is shown. Because the RCY for [⁶⁴Cu]Cu-FH (68.1%) was lower than that seen with [⁹⁰Y]Y-FH or [¹⁷⁷Lu]Lu-FH (90.1% or 95.1% in Figure 3a), variants of the standard HIR procedure, the HRR-HIR and HV-HRR-HIR procedures, were developed. (D) RCP by SEC after reaction mixes from (C) was purified by SEC. (E) RCY by TLC for reaction mixes yielding [⁹⁰Y]Y-FH or [¹⁷⁷Lu]Lu-FH is shown. With TLC, radio-DFO complexes remain at the origin (arrows) while negatively charged radio-FH NPs move to the solvent front on our negative charged, cation exchange TLC plates. (F) RCP by TLC after reaction mixes from (E) was purified by SEC. (G) RCY by TLC for reactions yielding ^64CuCu-FH. (H) RCP by TLC after reaction mixes from (G) was purified by SEC.