Toward the Development of Biomimetic Polymers by Protein Immobilization: PEGylation of Insulin as a Model Reaction

Abstract

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.

Covalently immobilized biosignal molecule materials for tissue engineering

“…Some studies using the spacer chains were performed and the increase in the activity is reported. 26,[29][30][31][32]48 However, possible implications of steric hindrance in spacing growth factor from the substrate and the possible impact on growth factor binding to its cell surface receptor should be taken into consideration. In fact, the POE conjugation reduced the biological activity of insulin.…”

Enhanced osteogenic promotion around dental implants with synthetic binding motif mimicking bone morphogenetic protein (BMP)‐2

“…9,10 Much effort has been devoted to ensure the stable localization of bioactive molecules, using either physical adsorption or chemical crosslinking methods. 15,24 Physical adsorption using soluble growth factor may not be sufficient in promoting long-term implantation due to factors such as protein desorption and/or exchange with physiologic fluid contact. In contrast, chemical binding involves a covalent attachment of the target molecule to the solid surface, thereby resulting in irreversible binding with high levels of surface coverage, and making this approach more suitable.…”

Topical Insulin in Neurotrophic Keratopathy: A Review of Current Understanding of the Mechanism of Action and Therapeutic Approach

“…Currently, lubricating drops containing polyethylene glycol 400 are available as ready-to-use. Such solutions tend to be stable, have reduced risk of proteolysis, and have minimal potential for antigenicity and impact on the patient’s immune system [ 35 , 36 ]. Additionally, the polyethylene glycol in available lubricating drops can accelerate epithelial defect healing [ 37 ].…”