Toward the development of biomimetic polymers by protein immobilization: PEGylation of insulin as a model reaction

Many current tissue-engineering investigations aim at the rational control of cell adhesion and tailored composition of biomaterial surfaces by immobilizing various protein and peptide components, such as growth factors. As a step on the way to develop polymers that allow for such surface modifications, water-soluble polymers were used as model substances to examine reactions with proteins containing amine groups. Consequently, the uncommon PEGylation of insulin in aqueous buffers was used to characterize reaction products and simulate the intended immobilization step for surface modification. Amine reactive poly(ethylene glycol)s were synthesized and characterized by (1)H nuclear magnetic resonance and gel-permeation chromatography. Furthermore, the model protein insulin was characterized concerning its accessible amino groups, using a fluorescent dye (TAMRA-SE). The resulting reaction products were identified by reversed-phase high-performance liquid chromatography and electrospray mass spectrometry. After PEGylation with hydrolytically stable poly(ethylene glycol) succinimidyl ester, the obtained PEGylated insulin was investigated by gel filtration chromatography, indicating successful attachment of the hydrophilic polymer chains. Application of an aqueous PEGylation scheme opens the door to the immediate investigation of various growth factors in cell culture, allowing for direct assessment of biological activity after forming the polymer-protein constructs with regard to later immobilization on surfaces.