Winscan radio tlc software

Unless otherwise mentioned, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and used as received, and all instruments were calibrated and maintained in accordance with standard procedures. [⁸⁹Zr]Zr⁴⁺was produced at Memorial Sloan Kettering Cancer Center on a TR19/9 cyclotron (Ebco Industries Inc.) via the ⁸⁹Y(p,n)⁸⁹Zr reaction and purified to yield [⁸⁹Zr]Zr⁴⁺ with a specific activity of 196–496 MBq/mg. Activity measurements were made using a CRC-15R Dose Calibrator (Capintec). The radiolabeling of ligands was monitored using instant thin-layer chromatography paper (Agilent Technologies) and analyzed on a Bioscan AR-2000 radio-ITLC plate reader using Winscan Radio-TLC software (Bioscan Inc.). Radioactive samples from in vitro immunoreactivity assays and ex vivo biodistribution studies were counted on an Automatic Wizard gamma counter (Perkin Elmer). All in vivo experiments were performed under the guidelines enumerated by the Research Animal Resource Center and protocols approved by the Institutional Animal Care and Use Committee at Memorial Sloan Kettering Cancer Center, NY.

For a typical chelator-free ⁸⁹Zr labeling of cRGDY-PEG-C′ dots, 4 nmol of cRGDY-PEG-C′ dots was mixed with 1 mCi of ⁸⁹Zr-oxalate in HEPES buffer (pH 8) at 75 °C. The radiolabeling yield of cRGDY-PEG-C′ dots at 1, 30, 60, 120, and 240 min was monitored using salicylic acid impregnated instant thin-layer chromatography paper (ITLCSA) (Agilent Technologies), and analyzed either on a Bioscan AR-2000 radio-TLC plate reader using Winscan Radio-TLC software (Bioscan Inc., Washington, DC) or an Automatic Wizard^{2 (link)}γ-Counter (PerkinElmer). After incubation, 5 μL aliquots were withdrawn and mixed with 50 μL of EDTA (50 mM, pH 5–6) before analyzing by ITLC using EDTA (50 mM, pH 5–6) as a mobile phase solvent. Free ⁸⁹Zr formed an instantaneous complex with EDTA and eluted with the solvent front, while ⁸⁹Zr-labeled cRGDY-PEG-C′ dots remained at the origin. For more accurate quantification, strips were cut in half, and γ-ray emissions at 909 keV were counted on a calibrated γ-counter (PerkinElmer) using a dynamic energy window of 800–1000 keV. Similar procedures were introduced when studying the pH-, concentration-, and temperature-dependent chelator-free labeling of cRGDY-PEG-C′ dots. The specific activity of chelator-free ⁸⁹Zr-labeled cRGDY-PEG-C′ dots was found to be in the range of 100–500 Ci/mmol.

To study in vitro radio stability, both chelator-free and chelator-based ⁸⁹Zr-labeled cRGDY-PEG-C′ dot probes (∼100 μCi in 100 μL PBS) were kept in PBS (1×, 900 μL) and human serum (1×, 900 μL) at 37 °C under stirring at 650 rpm. Radiochemical purity was measured over a 5-day period by ITLC at various time points from the end of synthesis. The measurement was repeated three times at each time point. For in vivo radio stability, healthy female athymic nu/nu mice (6–8 weeks old, Taconic Farms Inc.) were injected with ∼200 μCi (∼7.4 MBq) of chelator-free (or chelator-based) ⁸⁹Zr-labeled cRGDY-PEG-C′ dots. Whole blood was collected at 2, 24, and 48 h postinjection, the plasma fraction (which contained >98% of the ⁸⁹Zr-labeled cRGDY-PEG-C′ dots) was separated from the whole blood at different postinjection time points by centrifugation at 8000 rpm for 10 min and used to test the radiopurity. The nonspecific association of ⁸⁹Zr-labeled cRGDY-PEG-C′ dots to the red blood cells was estimated to be less than 2%. The percentage of the intact ⁸⁹Zr-labeled cRGDY-PEG-C′ dots was then measured by using ITLC with the plates analyzed on a Bioscan AR-2000 radio-TLC plate reader using Winscan Radio-TLC software (Bioscan Inc., Washington, DC). The measurement was repeated three times at each time point.