Ar 2000 radio tlc plate reader

To label MSNs with ⁶⁸Ga, a chelator-free reaction method was used^{10 (link),22 (link)}. This approach results in the formation of a stable coordination complex between the radiometals and deprotonated Si–O–groups on the MSN surface First, 4.5 µg of MSNs, activated in ethanol overnight, were mixed with 1.5 ml of ⁶⁸GaCl₃ solution (~ 600 MBq) eluted with HCl (0.1 N), 30 µl of amonium hydroxide solution (Sigma-Aldrich, St Louis, MO, USA), and 100 µl of MES buffer (0.1 M, pH 7.3) to maintain a reaction pH of 7.3. The mixture was incubated at 75 °C for 15 min, and then centrifuged at 14,000 rpm to remove residual ⁶⁸Ga. Radiolabeling purity was analyzed using an AR-2000 radio-TLC plate reader (BioScan Inc., Washington, DC, USA). Before cell treatment, the MSNs were coated with a lipid bilayer using cationic liposome transfection agent (Lipofectamine 2000, Invitrogen, California, USA), as previsouly described^{10 (link)}.

All chemicals, unless otherwise noted, were purchased from Sigma-Aldrich, and used as received without further purification. Ultrapure water produced by a PURELAB Ultra system from ELGA was used throughout (18.2 MΩ cm). NMR spectra were acquired on a Bruker Avance III HD spectrometer operating at 600 MHz. The conjugates were purified by FPLC on an Äkta Pure 25 M chromatography system (GE Healthcare Life Sciences). MALDI-TOF mass spectra were acquired on an ultrafleXtreme instrument (Bruker Daltonics). Size-exclusion chromatography (SEC-HPLC) analyses were performed on a JASCO HPLC system LC-2000 analytical series equipped with a Superdex 200 5/150 GL column. Instant thin-layer chromatography (iTLC) was performed using sheets impregnated with salicylic acid (iTLC-SA; Agilent) eluted with 0.1 M EDTA, pH 5.0, and analysed on an AR-2000 radio-TLC plate reader (Bioscan Inc.). PET/CT scans were acquired on an Inveon animal PET scanner (Siemens Preclinical Solutions).

DFO-αCD133 or ^deglyDFO-αCD133 was radiolabeled
with [⁸⁹Zr]Zr⁴⁺ according to the standard published
protocols.^{18 (link)} In brief, each immunoconjugate
(0.5 mg) was
diluted in Chelex-treated PBS to a final concentration of 0.5 mg/mL.
[⁸⁹Zr]Zr⁴⁺ [92.5–370 MBq (2.5–10
mCi)] in 1.0 M oxalic acid was then diluted with Chelex-treated PBS,
and the solution pH was adjusted to 7.0–7.5 with 1.0 M Na₂CO₃ (final volume: 100 μL). After the bubbling
of CO₂ stopped, the ⁸⁹Zr solution was added
to the antibody solution, mixed thoroughly, and incubated on a ThermoMixer
for 15 min at 500 rpm and 37 °C. The progress of the reaction
was monitored via radio-instant thin layer chromatography (iTLC) with
an eluent of 50 mM ethylenediaminetetraacetic acid (EDTA), pH 5.0,
an AR-2000 Radio-TLC plate reader, and Winscan Radio-TLC software
(Bioscan, Inc.; Washington, DC, USA). Once the reaction reached completion,
free [⁸⁹Zr]Zr⁴⁺ was removed via size exclusion
chromatography. The radiochemical purity of the final radiolabeled
construct was assayed using radio-iTLC with an eluent of 50 mM EDTA,
pH 5.0.