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We have used the orthogonal carbodiimide condensation and copper-catalyzed azide-alkyne "click" cycloaddition (CuAAC) reactions to prepare self-assembled monolayers that present distinct peptides to stem cells in a bioinert background. The approach involved first forming mixed SAMs with three components: (i) an azide-terminated hexaethylene glycol alkanethiolate (HS-EG6-N3), (ii) a carboxylate-terminated hexaethylene glycol alkanethiolate (HS-EG6-COOH), and (iii) a triethylene glycol alkanethiolate (HS-EG3). An acetylene-bearing peptide and an amine-terminated peptide were then immobilized to these substrates using a "click" CuAAC reaction and a carbodiimide condensation reaction, respectively. Polarization-modulated infrared reflectance-absorbance spectroscopic analysis demonstrated formation of well-ordered, close-packed self-assembled monolayers (SAMs), chemoselective conjugation of amine-terminated peptides to surface carboxylate groups, and subsequent conjugation of acetylene-terminated peptides to the azide groups on SAMs. Varying the mole fraction of HS-EG6-N3, HS-EG6-COOH, and HS-EG3 during SAM formation allowed for control over the densities of each peptide on the substrate. Substrates presenting varying surface densities of RGESP (a nonfunctional peptide), RGDSP (a cell adhesion peptide), or TYRSRKY (a heparin/heparan sulfate-binding peptide) were then used to characterize the relationship between peptide surface density and human mesenchymal stem cell (hMSC) adhesion. Results demonstrate that RGESP does not influence RGDSP-mediated adhesion of hMSCs, which indicates that a second peptide with distinct bioactivity can be immobilized alongside RGDSP to characterize the influence of two peptides on hMSC behavior. Our results also demonstrate that RGDSP and TYRSRKY act synergistically to promote hMSC adhesion in the absence of serum. Interestingly, heparin sequestered by TYRSRKY inhibits cell adhesion on substrates presenting RGDSP = 0.1% and > or = 0.1% TYRSRKY or RGDSP = 1% and > or = 0.5% TYRSRKY. Taken together, these results indicate that two peptides can be controllably presented to stem cells on the same otherwise bioinert SAM substrate, and that multiple, distinct extracellular moieties act in concert to regulate hMSC adhesion.

Self-assembled monolayers (SAMs) of alkanethiolates on gold have become an important tool for probing cell-material interactions. Emerging studies in stem cell biology are particularly reliant on well-defined model substrates, and rapid, highly controllable fabrication methods may be necessary for characterizing the wide array of stem cell-material interactions. Therefore, this study describes a rapid method for creating SAM cell culture substrates with multiple discrete regions of controlled peptide identity and density. The approach uses a NaBH(4) solution to selectively remove regions of bioinert, hydroxyl-terminated oligo(ethylene glycol) alkanethiolate SAM and then locally replace them with mixed SAMs of hydroxyl- and carboxylic acid-terminated oligo(ethylene glycol) alkanethiolates. The cell adhesion peptide Arg-Gly-Asp-Ser-Pro (RGDSP) was then covalently linked to carboxylic acid-terminated mixed SAM regions to create cell adhesive environments within a bioinert background. SAM preparation and peptide immobilization were characterized using polarization modulation-infrared reflection-absorption spectroscopy (PM-IRRAS), as well as assays to monitor conjugation of a fluorescently labeled peptide. This "localized SAM replacement" method was achieved using an array of microchannels, which facilitated rapid and simple processing. Results indicate that immobilized RGDSP promoted spatially localized attachment of human mesenchymal stem cells (hMSCs) within specified regions, while maintaining a stable, bioinert background in serum-containing cell culture conditions for up to 14 days. Cell attachment to patterned regions presenting a range of cell adhesion peptide densities demonstrated that peptide identity and density strongly influence hMSC spreading and focal adhesion density. These substrates contain discrete, well-defined microenvironments for stem cell culture, which could ultimately enable high-throughput screening for the effects of immobilized signals on stem cell phenotype.

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 bio-inert background. The surface preparation approach involved first forming mixed SAMs with an azide-terminated hexaethylene glycol alkanethiolate (HS---EG6---N3) and a triethylene glycol alkanethiolate (HS---EG3), then using the CuAAC reaction to immobilize an alkyne-terminated peptide. The mixed SAMs were classified as bio-inert, as SAMs comprised of 10 mole percent HS---EG6---N3 and 90 mole percent HS---EG3 showed minimal non-specific 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 ∼ 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 non-specifically adsorbed serum proteins. Results indicate that an RGDSP intermolecular spacing of 36 nm or less (≥ 0.01 mole percent on the surface) is sufficient for hMSC adhesion and a spacing of 11 nm or less (≥ 0.05 mole percent 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 bio-inert SAMs, and that the resulting SAMs can be used to study the dependence of peptide density on stem cell behavior.