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.