Conjugation of the ATF24 peptide with DSPE-PEG2000-COOH

The uPAR targeting peptide, ATF_24, was conjugated with DSPE-PEG₂₀₀₀-COOH by a dehydration condensation reaction to obtain the targeting compound, DSPE-PEG₂₀₀₀-ATF₂₄. Briefly, the ATF₂₄ peptide, DSPE-PEG₂₀₀₀-COOH, and hexafluorophosphate benzotriazole tetramethyl uronium were dissolved in newly distilled dimethylformamide (molar ratio: 1:3:3), and then a 10-fold higher amount of diisopropylethylamine was immediately added. After 50 min of incubation at room temperature under moderate stirring, the reaction mixture was dialyzed [molecular weight (MW) cutoff = 3,500 Da] against deionized water to remove the unconjugated peptide and reaction medium. The final solution was lyophilized and stored at −20 °C until use. Liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS) (AB Sciex, Redwood City, CA, USA) was used to investigate the MW of the ATF₂₄ peptide. The MW of DSPE-PEG₂₀₀₀-ATF₂₄ was determined by a matrix assisted laser desorption/ionization-time of flight mass spectrometer (MALDI-TOF-MS) (Bruker Daltonics, Hamburg, Germany)²⁴.