Synthesis of APTEDB-conjugated PEG2000-DSPE

EDB-FN-specific aptamer-like peptide (APT_EDB; Anygen) containing an additional cysteine residue was dissolved in dimethyl sulfoxide (Sigma-Aldrich), and Mal-PEG₂₀₀₀-DSPE was dissolved in chloroform (Sigma-Aldrich). The conjugation reaction was carried out at an APT_EDB:Mal-PEG₂₀₀₀-DSPE molar ratio of 1:2 under inert conditions for 12 h at ambient temperature ¹⁶. APT_EDB-conjugated PEG₂₀₀₀-DSPE (APT_EDB-DSPE) was purified by reversed-phase high-performance liquid chromatography, and the conjugation efficiency was determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. To remove unconjugated peptides, dialysis was carried out using a dialysis membrane (Spectrum Chemical, NJ, USA) with a molecular weight cutoff of 3.5 kDa. After 48 h, the conjugate was lyophilized.