Hydrogel Precursor Solution Synthesis

For hydrogel precursor solution preparation, peptides containing alloxycarbonyl (alloc)–protected lysines, K(alloc) that provides a reactive alkene, were synthesized by solid-phase peptide synthesis and characterized using established methods as previously described (30 (link), 42 (link)). Briefly, the pendant integrin-binding peptide sequence K(alloc)G(POG)₃-POGFOGERG-(POG)₄-G (GFOGER) and enzymatically degradable linker peptide K(alloc)GGPQG↓IWGQGK(alloc) were synthesized using a Liberty Blue Automated Microwave Peptide Synthesizer (CEM, Matthews, NC). The GFOGER sequence was synthesized using a high-swelling ChemMatrix resin (Protein Technologies), and the linker peptide was synthesized on rink amide 4-Methylbenzhydrylamine (MBHA) resin (Novabiochem). For the peptide collection from resin, a cleavage solution was prepared using 95% trifluoroacetic acid (Arcos Organics), 2.5% triisopropylsilane (Arcos Organics), and 2.5% water (all percentages v/v) supplemented with phenol (25 mg ml⁻¹) (Research Products International), and then incubated with a peptide-containing resin for 4 hours. After cleavage from the resin, all peptides were collected by precipitation in cold diethyl ether (9× excess volume) overnight at 4°C and purified by reverse-phase high-performance liquid chromatography (XBridge BEH C18 OBD 5-μm column; Waters, Milford, MA) with a linear water-acetonitrile (ACN) gradient (water: ACN 95:5 to 45:5; 1.17% change in water per minute). Purified peptides were lyophilized and their molecular weights confirmed via mass spectrometry (30 (link)). Twenty-kilodalton four-arm polyethylene glycol tetra thiol (PEG-4SH) was either synthesized in the laboratory (42 (link)) or purchased (JenKem Technology, Plano, TX), breaking any disulfides before use by overnight treatment with tris(2-carboxyethyl)phosphine (350 mg per 1 g of PEG-SH in ≈30 ml) followed by dialysis (molecular weight cutoff of 1 kDa, Spectrum Laboratories, for 24 hours against deionized water at pH 4) and lyophilization. Linker and pendant peptides and PEG-4SH were dissolved in sterile PBS (Invitrogen) supplemented with 1% PS (Invitrogen) and fungizone (0.5 μg ml⁻¹, Invitrogen). Hydrogel precursor solution was prepared by mixing stock solutions for achieving 6% PEG-4SH by weight (wt %), 2 mM pendant peptide, and the balance of linker peptide (1:1 SH:alloc).