High density binding of proteins and peptides to poly(D,L-lactide) grafted with polyacrylic acid.

The use of graft polymers for the functionalisation of biomaterial surfaces is already widespread. We investigated the adsorptive and covalent binding of a variety of proteins and peptides to poly(D,L-lactide) grafted with polyacrylic acid. Covalent attachment was achieved through coupling of amino groups of the protein/peptide to the carboxyl groups of the graft polymer by using a water-soluble carbodiimide and N-hydroxysuccinimide. Binding densities were determined by automated amino acid analysis after acid hydrolysis of both the poly(D,L-lactide) and the adsorbed and covalently bound proteins. Experiments in the absence and presence of the coupling reagents allow to discriminate between adsorptive and covalent binding. Although the adsorptivc binding is quite substantial in absolute terms, the amount of adsorbed protein is relatively low as compared to the total amount of bound protein. Total binding densities of 20-30 microg/cm2 can easily be achieved. Depending on the concentration and on the properties of the proteins and peptides, between 5% and 80% of the totally bound protein may be physically adsorbed. Densities expressed in molecules/10 nm2 vary from 0.5 molecule fibronectin to 2,000 laminin-peptide molecules: their binding densities clearly correlate with their respective molecular masses. Obviously, the binding densities are governed by their individual three-dimensional space requirements rather than the density of the available carboxyl groups. From the number of carboxyl groups/10 nm2 (18,000-30,000 COOH/10 nm2) the average length of the acrylic acid graft polymer molecules was estimated. Based on the assumption that about 10 copolymer chains can be accommodated on 10 nm2, the average length of the polymer chains, which corresponds to the thickness of the graft phase, is estimated to be 0.5-1 microm. The organisation of the proteins and peptides within the polyacrylic acid phase was further investigated by experiments in which a protein (BSA) and a peptide (Val-Lys) were allowed to react in either a singular, a consecutive or a simultaneous way. Together with XPS and IR-ATR surface characterisation experiments a three-dimensional picture of the arrangement of the immobilised proteins and peptides within the graft polymer phase emerges.