Itlc sg strip

The complexation of ⁸⁹Zr with 1 was achieved by reacting 10 µg (10 µL, 1.0 mg/mL in water) of 1 with an aliquot of ⁸⁹Zr(Ox)₂ (22.2 MBq) that was diluted in 100 µL of water and pH adjusted to 7-7.5 using 1 M Na₂CO₃. The reactions were incubated at 24°C for 15 min in a thermomixer (550 rpm). Formation of ⁸⁹Zr-1, was monitored by radio-TLC using Varian ITLC-SG strips and 50 mM DTPA (pH 7) as the mobile phase. In this system, free ⁸⁹Zr forms a complex with DTPA and is eluted with the solvent front, while ⁸⁹Zr-1 remains at the origin. The identity of each radioactive complex was further confirmed by comparing its radio-HPLC elution profile to the UV-HPLC spectrum of ^NatZr-1.

In vitro stability was carried out by adding 10 µL of each ⁸⁹Zr-labeled complex (1.85 MBq) to 500 µL DTPA (50 mM, pH 7), or human serum. The solutions (n = 12) were incubated at 37°C for 7 days and were analyzed daily for 1 week by radio-TLC using Varian ITLC-SG strips and 50 mM DTPA (pH 7) as the mobile phase and gamma counting using an energy window of 500-1500 keV and standard protocols 19 (link).

⁸⁹Zr-oxalic
acid solution (1M) was purchased from PerkinElmer (Waltham, US). ZEGFR:2377
targeting bioconjugates were labeled with zirconium-89 according to
following protocol, corresponding to a modified variant of Vosjan
and co-workers.^{46 (link)89}Zr solution
(8.7 μL, 10–12 MBq) was neutralized by adding 8.3 μL
of Na₂CO₃ (1M) and incubated for 3 min at RT.
Hereafter, 66.7 μL of HEPES buffer (0.5M; pH 6.98) was added
to the radionuclide containing solution. After subsequent addition
of 20 μL of bioconjugate solution (1 μg/μL in H₂O/EtOH 90/10 v/v), the resulting mixture was incubated up
to 120 min at 28 and 85 °C, respectively. Radiolabeling was monitored
by radio instant thin layer chromatography (radio-ITLC) using silica gel-impregnated glass microfiber sheets (ITLC-SG
strips, Varian, Lake Forest, CA) as stationary and 0.2 M citric acid
(pH 5) as mobile phase. Distribution of the radioactivity among the
strips was measured on Cyclone Phosphor Storage Screen using OptiQuant
software for data processing (both Packard Instrument Company, Meriden,
CT, US) as well as Fujifilm Bioimaging Analyzers (BAS) 1800II using
MultiGauge V3.0 analysis software (both Fujifilm, Tokyo, Japan). Radiolabeled
bioconjugates were purified for further studies using phosphate buffer
saline pre-equilibrated NAP-5 size exclusion columns (GE Healthcare,
Uppsala, Sweden) according to manufacturer’s protocol.

[⁸⁹Zr]Zr-oxalate solution
was neutralized with sodium carbonate 1 M to pH = 7–7.5. 4HMS
and DFO were prepared, respectively, in ethanol and water (1.1 and
1 mg/mL). An aliquot of 4HMS or DFO (5 μL, 7.65 nmol) was radiolabeled
with neutralized [⁸⁹Zr]Zr-oxalate (30 MBq, 200 μL)
in either water or 0.9% NaCl at room temperature for 5–10 min
with a total volume 205 μL. The radiolabeling was monitored
by radio-TLC using Varian ITLC-SG strips and 100 mM DTPA (pH 7) as
the mobile phase. In this system, free ⁸⁹Zr forms a complex
with DTPA and eluted with the solvent front, while the ⁸⁹Zr-ligand complex remained at the origin (Figure S20). The identity of the [⁸⁹Zr]Zr-4HMS radioactive
complex was further confirmed by comparing its radio-HPLC elution
profile to the ultraviolet-HPLC spectrum of ^natZr-4HMS
(Figure S21).