Oligopeptides are versatile cell modulators resembling pleiotropic activities of ECM proteins and growth factors. Studying the role of cell-instructive peptide signals within 3D scaffolds, yet poorly known, requires effective approaches to introducing bioactive sequences into appropriate materials. We synthesized RGD and GHK motif based peptides 1 and 2 linked to the terminal adamantyl group (Ad) and their fluorescent derivatives 3 and 4. Poly(hydroxyethyl methacrylate) (pHEMA) cryogels with additional PEG/β-cyclodextrin (CD) units were prepared as an inert macroporous scaffold capable to bind the adamantylated peptides via affinity CD-Ad complexation. According to toluidine blue staining, the CD moieties were effectively and stably incorporated in the pHEMA cryogels at nanomolar amounts per milligram of material. The CD component gradually increased the thickness and swelling ability of the polymer walls of cryogels, resulting in a noticeable decrease in macropore size and modulation of viscoelastic properties. The labeled peptides exhibited fast kinetics of specific binding to the CD-modified cryogels and were simultaneously immobilized by coincubation. The peptide loading approached ca. 0.31 mg per cm2 of cryogel sheet. A well-defined mitogenic effect of the immobilized peptides (2 < 1≪ 1 + 2) was revealed toward 3T3 and PC-12 cells. The synergistic action of RGD and GHK peptides induced a profound change in cell behavior/morphology attributed to a growth-factor-like activity of the composition. Altogether, our results provide an effective procedure for the preparation of CD-modified pHEMA cryogels and their uniform in situ functionalization with bioactive peptide(s) of interest and an informative study of cellular responses in the functionalized scaffolds.
Keywords: GHK; RGD; affinity immobilization; bioactive peptide motifs; cell responses; cryogels; cyclodextrin-adamantane inclusion complex; extracellular matrix; growth factors; poly(hydroxyethyl methacrylate).