Abstract
We have used a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) "click" reaction to prepare self-assembled monolayers (SAMs) presenting the cell adhesion peptide Arg-Gly-Asp-Ser-Pro (RGDSP) in a bioinert background. The surface preparation approach involved first forming mixed SAMs with an azide-terminated hexa(ethylene glycol) alkanethiolate (HS---EG6---N3) and a tri(ethylene glycol) alkanethiolate (HS---EG3), then using the CuAAC reaction to immobilize an alkyne-terminated peptide. The mixed SAMs were classified as bioinert, as SAMs comprising 10 mol % HS---EG6---N3 and 90 mol % HS---EG3 showed minimal nonspecific protein adsorption in solutions of 1 mg/mL lysozyme or 10% fetal bovine serum. The reaction between an acetylene-terminated peptide and an azide-terminated SAM proceeded rapidly and quantitatively in the presence of a Cu(I)-TBTA complex, displaying pseudo-first-order kinetics with a rate constant of approximately 0.2 min(-1). Varying the ratio of HS---EG6---N3 to HS---EG3 during SAM formation allowed for control over the density of azide and, in turn, the density of RGDSP on the substrates. These substrates were therefore used to study the detailed relationship between RGDSP surface density and human mesenchymal stem cell (hMSC) adhesion, spreading, and focal adhesion complex formation, without interference from nonspecifically adsorbed serum proteins. Results indicate that an RGDSP intermolecular spacing of 36 nm or less (>or=0.01 mol % on the surface) is sufficient for hMSC adhesion and a spacing of 11 nm or less (>or=0.05 mol % on the surface) is sufficient for cell spreading and focal adhesion complex formation. In total, our results demonstrate that CuAAC is a suitable mechanism for conjugating peptides to otherwise bioinert SAMs and the resulting SAMs can be used to study the dependence of peptide density on stem cell behavior.