PREPARATION OF CELL-AFFINITY ADSORBENTS BY IMMOBILIZATION OF PEPTIDESTO SEPHADEX G-10

In this study, affinity adsorbents for the binding of activated blood platelets and endothelial cells were prepared from Sephadex G-10 by im mobilization of peptides, derived from the cell-adhesive amino acid se quence RGD (arginine-glycine-aspartic acid). Derivatization of Sephade x G-10 was accomplished by sequential coupling of specific dipeptides (RG and DV) (V = valine) and by coupling of the RGD-containing hexapep tide GRGDSP (S = serine, P = proline). Two types of gel were prepared by sequential, coupling, designated as G10 (acetone) and G10 (dimethyl formamide) (G10 (DMF)), containing peptides which had been coupled to the outer side of the beads and throughout the porous beads, respectiv ely. The binding capacity of the prepared Sephadex-derivatives amounte d up to 2 x 10(9) human blood platelets per millilitre GRGDV-Sephadex at immobilized peptide concentrations, that were in the nanomole range (per millilitre packed gel) and which differed a factor 10 between G1 0 (acetone) and G10 (DMF). In a second series of experiments, differen t amounts of the hexapeptide GRGDSP were coupled to carboxylated Sepha dex G-10 and carboxylated Sepharose CL 6B. The binding of human umbili cal vein endothelial cells to the resulting materials was studied. Up to 10(6) endothelial cells attached per ml GRGDSP-derivatized hydrogel at peptide concentrations of 15 nmol GRGDSP/ml Sephadex and at +/-300 nmol GRGDSP/ml Sepharose. Substitution of the arginine residue of the RGD-sequence by glutamine abolished the cell-binding activity of the immobilized peptide towards activated blood platelets but not towards endothelial cells. From the results of this study it can be concluded that small peptides can be coupled to the outer side of the porous Sep hadex beads, resulting in high effective ligand densities for cell-aff inity applications. In this respect, Sephadex G-10, derivatized accord ing to 'the acetone method', is a good alternative for polystyrene and other solid phase materials.