Covalent core-radiolabeling of polymeric micelles with 125I/211At for theranostic radiotherapy

Astatine-211 (211At) is one of the most promising α-emitters for targeted alpha therapy, especially of cancer metastases. However, the lack of a stable isotope, frequent in vivo deastatination, and limited radiochemical knowledge makes it challenging to apply. Here, we report a new strategy for radiolabeling the lipophilic core of polymeric micelles (PMs) with covalently bound 211At. The PMs were radiolabeled via either an indirect synthon-based method or directly on the amphipathic block copolymer. The radiochemistry was optimized with iodine-125 (125I) and then adapted for 211At, enabling the use of both elements as a potential theranostic pair. PMs that were core-radiolabeled with both 125I or 211At were prepared and characterized, based on a PEG(5k)-PLGA(10k) co-polymer. The stability of the radiolabeled PMs was evaluated in mouse serum for 21 h, showing radiochemical stability above 85%. After in vivo evaluation of the 211At- labeled PMs, 4-5 % ID/g of the 211At could still be detected in the blood, showing a promising in vivo stability of the PMs. Further, 211At-labeled PMs accumulated in the spleen (20-30 %ID/g) and the liver (2.5- 5.5 %ID/g), along with some detection of 211At in the thyroid (3.5-9 %ID/g). This led to the hypothesis that deastatination takes place in the liver, whereas good stability of the 211At core-radiolabel was observed in the blood.


S2 -Radio-TLCs from the indirect labeling of 211 At-PMs
Every step of the indirect labeling procedure of PEG(5k)-PLGA(10k) with 211 At was followed via radio-TLC.
Labeling of the stannyl-benzoate (1) with NIS could achieve a RCC of >95 % (Figure S2 A) and results in 211 At-benzoat (2). The conjugation of this 211 At-benzoat (2) with PEG(5k)-PLGA(10k)-NH 2 could be achieved after 3 h and according to radio-TLC, the RCC was 78 % (Figure S2 B). The polymer was then purified via centrifuge filter and analyzed via radio-TLC to follow the efficiency of the purification method ( Figure S2 C and S2 D).

S3 -Cryo-TEM picture of the 211 At-PMs
After the 211 At in the 211 At-PMs was decayed, cryo-TEM images were obtained. To that end, the 3 µl of sample in PBS were placed on 300 mesh Cu TEM grids with a holey carbon support film, blotted and plunge frozen in liquid nitrogen using a FEI Vitrobot mark IV. Frozen grids were then imaged on a FEI Tecnai T20 operated at 200 kV in low dose mode and images acquired using a FEI High-Sensitive (HS) 4k x 4k Eagle camera.

S4 -PD 10 analysis of the finally prepared polymeric micelles
The radio-halogenated PMs with 211 At and 125 I were analyzed via size exclusion chromatography and each fraction was measured on the gamma counter to see if any radioactivity could be separated from the PMs. As a control, free 125 I and 211 At were analyzed on the same cartridge. In all cases, the majority of the radioactivity (80-90 %) was detected in the PM fractions (fractions 2-6). The remaining activity appears to be tailing of the PMs, which allows the conclusion of successful labeling of the PMs with 125 I and 211 At. fractions were collected and the radioactivity measured on a well-counter. One fraction corresponded to 1 mL. The radio-halogenated PMs eluted in fraction 2-6, and the majority of the radioactivity was found in fraction 4 and 5. As controls, free 211 At or 125 I were mixed with non-radiolabeled PMs and also Supporting Information separated with the PD-10, were the majority of the 211 At/ 125 I could be found in fraction 7-12. This showed that the association between radionuclide and PM is not non-specific.

S5 -Stability study of 125 I-PMs and 211 At-PMs in PBS
The stability of the indirectly labeled 125 I-PMs and 211 At-PMs and the directly labeled 125 I-PMs was evaluated after incubation in PBS at 37 °C for 4 h (Figure S5 A) and 24 h (Figure S5 B). Free 211 At and 125 I were separated from the PMs via centrifuge filter, where the radio-halogenated PMs remain in the filter and free 211 At/ 125 I is seen in the combined washing fractions (filtrate). As described in more details in the main manuscript, not 100% of the radioactivity was detected after the separating procedure. Some of the radioactive material adsorbed to the filter and was, therefore, not detected. After 4 h, 91 ± 6% of the 125 I was found in the PM fraction, which dropped to 86 ± 6% within the next 20 h for the indirectly labeled PMs. For the directly labeled PMs, the amount of 125 I decreased from 87 ± 3% to 81 ± 7% after 24 hours. This again supported that the direct labeling is less stable, possibly due to free 125 I trapped in the core of the PMs.