Automatic wizard2 link γ counter

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 After incubation, 5 μL aliquots were withdrawn and mixed with 50 μL of EDTA (50 mM, pH 5–6) before analyzing by ITLC using EDTA (50 mM, pH 5–6) as a mobile phase solvent.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.

⁸⁹Zr 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 with a specific activity of 5.28–13.43 mCi/μg (470–1195 Ci/mmol) of zirconium.^{56 (link)} Activity measurements were performed using a CRC-15R Dose Calibrator (Capintec). For the quantification of activities, experimental samples were counted on an Automatic Wizard^{2 (link)} γ-Counter (PerkinElmer). All in vivo experiments were performed according to protocols approved by the Memorial Sloan Kettering Institutional Animal Care and Use Committee. A purity of greater than 95% was confirmed using radio-TLC for all of the ⁸⁹Zr-labeled DFO-αMSH-PEG-Cy5-C′ dots.

⁸⁹Zr 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 with a specific activity of 5.28–13.43 mCi/μg (470–1195 Ci/mmol) of zirconium.^{40 (link)} Activity measurements were performed using a CRC-15R Dose Calibrator (Capintec). For the quantification of activities, experimental samples were counted on an Automatic Wizard^{2 (link)}γ-Counter (PerkinElmer). All in vivo experiments were performed according to protocols approved by the Memorial Sloan Kettering Institutional Animal Care and Use Committee. A purity of greater than 95% was confirmed using radio-TLC for all of the ⁸⁹Zr-labeled cRGDY-PEG-C′ dots.

Following the terminal PET imaging timepoint (144 h p.i.), the mice were euthanized via CO₂ asphyxiation followed by cervical dislocation. The 15 most relevant organs were collected, rinsed in water, dried, weighed, and quantified using an Automatic Wizard^{2 (link)} γ-counter calibrated for ⁸⁹Zr (PerkinElmer). The counts/minute in each tissue was background- and decay-corrected to the start of the activity measurement. The %ID/g for each sample was calculated by normalization to the total injected activity.