Surface specific peptide immobilization on radiografted polymers as potential screening assays for antiangiogenic immunotherapy

Abstract

Angiogenesis is a key process of cancer development and metastasis. It’s inhibition is an important and promising strategy to block tumor growth and invasion. One of these approaches, based on antiangiogenic immunotherapy, is the recognition of a specific region of an angiogenic growth factor, called VEGF-A, by monoclonal antibodies. Thus, we aimed to design a novel assay to screen potential monoclonal antibodies directed against VEGF-A. In a first approach, we chose to perform covalent coupling of angiogenesis active cyclopeptides onto biocompatible thermoplastic transparent PVDF films and to fully characterize the chemical structure, the surface state and the biochemical properties of the synthesized devices. Electron beam radiation created radical sites on PVDF films without adding any toxic chemicals. These primary radicals and some induced peroxides were used as initiators for acrylic acid polymerization. Under our experimental conditions, surface grafting was favoured. Functionalization of PVDF-g-PAA films with peptides via a spacer arm was possible by performing two subsequent coupling reactions. EDC was used as coupling agent. Spacer arm saturation of the film surface was achieved for 25 mol% yield meaning that one spacer arm on four carboxylic acids were covalently bound. Peptide immobilization resulted in binding 10 times less leading to a final 3 mol% yield. Binding densities are governed by their individual space requirements. Each chemical step has been followed by FTIR in ATR mode, NMR using HR MAS technique and XPS. From XPS results, a layer of peptide covered PVDF-g-PAA film surface. The amounts of covalently immobilized peptide were determined using indirect UV spectroscopy on supernatant reaction solution. Yields were correlated with high resolution NMR results. The peptide/antibody recognition validated our system showing the conservation of peptide tridimensional structure with a positive response to specific antibodies. Because of the covalent protein linkage to PVDF films, a simple cleaning with immunoaffinity chromatography buffer allows the films to be reused.