Dual-Functional Nitric Oxide-Releasing Peptide Amphiphiles

Two types of PAs; PA-YIGSR [CH₃(CH₂)₁₄CONH-GTAGLIGQ-YIGSR] and PA-KKKKK [CH₃(CH₂)₁₄CONH-GTAGLIGQ-KKKKK], were prepared using the Fmoc chemistry in the Advanced Chemtech Apex 396 peptide synthesizer (AAPPTec, Louisville, KY, USA) and subsequently alkylated at the N-termini with palmitic acid by a manual coupling reaction for 24 hours at room temperature [60 (link)]. To alkylate with palmitic acid, a mixture of o-benzotriazole-N, N, N, N'-tetramethyluronium hexafluoro phosphate, di-isopropyl-ethylamine, and dimethylformamide was used, cleavage and deprotection were achieved using a mixture of trifluoroacetic acid, deionized water, triisopropylsilane, and anisole (40:1:1:1) for 3 hours at room temperature. The PAs precipitated in cold ether were lyophilized and characterized by matrix-assisted laser desorption ionization time of flight mass spectrometry. PA-YIGSR was composed of an endothelial cell adhesive ligand (YIGSR) coupled with a matrix metalloprotease-2 (MMP-2) degradable sequence (GTAGLIGQ) to form PA-YIGSR. PA-KKKKK contained a NO donor poly-lysine (KKKKK) linked to the MMP-2 degradable sequence, forming PA-KKKKK. A mixture of PA-YIGSR and PA-KKKKK at a 9:1 molar ratio was reacted with NO gas to generate PA-YK-NO [47 (link)]. For gelation process, 50 μL of a 2% wt stock PA-YK-NO solution was mixed with 15 μL of calcium chloride and 25 μL of phosphate-buffered saline (PBS) and incubated at 37°C for 30 min.