Rp hplc

RGD-Aoc-(Arg¹¹)CCMSH and RGD-PEG₂-(Arg¹¹)CCMSH peptides were labeled with ^99mTc via a direct reduction reaction using SnCl₂. Briefly, 10 µL of 1 mg/mL SnCl₂ in 0.1 M HCl, 40 µL of 0.5 M NH₄OAc (pH 5.2), 100 µL of 0.2 M Na₂tartate (pH 9.2), 100 µL of fresh ^99mTcO₄⁻ solution (37–74 MBq), and 10 µL of 1 mg/mL of each peptide in aqueous solution were added into a reaction vial and incubated at 25 °C for 20 min to form ^99mTc-labeled peptide. Each ^99mTc-peptide was purified to a single species by Waters RP-HPLC (Milford, MA) on a Grace Vydac C-18 reverse phase analytic column (Deerfield, IL) using a 20-min gradient of 16–26% acetonitrile in 20 mM HCl aqueous solution at a flow rate of 1 mL/min. Each purified peptide was purged with N₂ gas for 20 min to remove the acetonitrile. The pH of final peptide solution was adjusted to 7.4 with 0.1 N NaOH and sterile normal saline for animal studies.

⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex were prepared as described in our previous publication (14 (link)). The proposed schematic structures of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex are shown in Figure 1. Briefly, 10 μL of ⁶⁴CuCl₂ (37–74 MBq in 0.05 M HCl aqueous solution), 10 μL of 1 mg/mL peptide aqueous solution, and 200 μL of 0.5 M NH₄OAc (pH 5.4) were added into a reaction vial and incubated at 75 °C for 1 h. After incubation, 10 μL of 0.5% EDTA aqueous solution was added to scavenge potentially unbound ⁶⁴Cu²⁺. ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex complexes were purified by Waters RP-HPLC (Milford, MA) on a Grace Vydac C-18 reverse phase analytical column (Deerfield, IL) with a flow rate of 1 mL/min. A 20 min gradient of 20–30% acetonitrile in 20 mM HCl aqueous solution was used for ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex, whereas a 20 min gradient of 24–34% acetonitrile in 20 mM HCl aqueous solution was utilized for ⁶⁴Cu-NOTA-AocNle-CycMSH_hex. Each purified peptide solution was purged with N₂ gas for 15 min to remove the acetonitrile, then adjusted to pH 7.4 with 0.1 M NaOH and sterile saline for animal studies.