Dual-Functionalized Nanostructured Biointerfaces by Click Chemistry

Abstract

The presentation of biologically active molecules at interfaces has made it possible to investigate the responses of cells to individual molecules in their matrix at a given density and spacing. However, more sophisticated methods are needed to create model surfaces that present more than one molecule in a controlled manner in order to mimic at least partially the complexity given in natural environments. Herein, we present dual-functionalized surfaces combining quasi-hexagonally arranged gold nanoparticles with defined spacings and a newly developed PEG-alkyne coating to functionalize the glass in the intermediate space. The PEG-alkyne coating provides an inert background for cell interactions but can be modified orthogonally to the gold nanoparticles with numerous azides, including spectroscopically active molecules, peptides, and biotin at controlled densities by the copper(I)-catalyzed azide alkyne click reaction. The simultaneous presentation of cRGD on the gold nanoparticles with 100 nm spacing and synergy peptide PHSRN in the space between has a striking effect on REF cell adhesion; cells adhere, spread, and form mature focal adhesions on the dual-functionalized surfaces, whereas cells cannot adhere on either monofunctional surface. Combining these orthogonal functionalization methods creates a new platform to study precisely the crosstalk and synergy between different signaling molecules and clustering effects in ligand–receptor interactions.

Rapid Reversible Photoswitching of Integrin‐Mediated Adhesion at the Single‐Cell Level

“…The molar ratio of c(RGDfK)-azobenzene and PEG2000 was adjusted to 1:99, which has recently been reported to offer adequate adhesion conditions for fi broblast cells. [ 21 ] It corresponds to a c(RGDfK)-azobenzene packing density of ≈0.5 molecules/nm 2 , as determined with UV-vis spectroscopy. Figure 1 B illustrates the working hypothesis of the mixed c(RGDfK)-azobenzene/PEG2000 monolayer.…”

Bioactive compounds immobilized on Ti and TiNbHf: AFM-based investigations of biofunctionalization efficiency and cell adhesion

“…The PHSRN sequence is needed to achieve full adhesive activity for the α 5 β 1 integrin receptor. Previous research has shown that high affinity binding requires this synergy site to be engaged [57,58], and that availability of both the synergy site and RGD has significant impact on cell adhesion [38,59]. Hence, CAS is clearly superior to RGD in initial cell adhesion.…”

“…Although the binding affinity of RGD has strongly been improved by cyclization of the RGD sequence [34,35], which is realized by adding flanking residues and constraining the conformation of the RGD motif to a loop [36,37], the RGD binding sequence does not recapitulate the full functionality of ECM proteins. Native proteins contain multiple cell-binding sequences, which allow dynamic and simultaneous binding to cellular receptors and positively trigger cell adhesive events [38].…”

Multifunctional biomaterial coatings: synthetic challenges and biological activity

“…They immobilized the integrin binding motifs PHSRN and RGD in one molecule on titanium and could show beneficial influence on osteoblast-like cell behavior over a mixture of both peptides. Defined spatial distributions could be also realized by a patterned surface displaying PHSRN and RGD, which also resulted in a positively synergic effect on cell fate (Schenk et al, 2014).…”

“…While employing a bioinert background by PEG chains, cell-attracting cRGD was immobilized on gold nanoparticles and PHSRN was coupled by CuAAC to the silanized glass surface. The defined distribution of these peptides had a pronounced impact on cell number, area and the generation of focal contacts (Schenk et al, 2014). In Figure 5E it is schematically shown how the requirements of cardiovascular biomaterials can be implemented in an experimental setup.…”