Mussel-inspired adhesive epidermal growth factor for biological surface modification

摘要

Introduction: Growth factor, as a fundamental requirement in tissue engineering, plays important roles in the regulation of a variety of cellular processes. It has been demonstrated that the immobilized growth factors on the material surfaces can stimulate the cognate receptors in the cell membrane and trigger the signal transduction. Although types of growth factors with binding ability to specific substrates were developed, simple and versatile strategies to modify growth factors for surface modification of multiple classes of materials are essential. Mere inspired by underwater adhesive protein secreted by mussel, we incorporated the binding peptide sequence carrying 3,4-dihydroxyphenylalanine (DOPA) to the active sequence of epidermal growth factor (EGF). The binding affinity of the constructed growth factor (EGF-DOPA) to typical organic and inorganic materials and its biological activities were investigated. Materials and Methods: A peptide synthesizer ABI 433A was employed to synthesize the active sequence of EGF and the adhesive peptide carrying DOPAs and lysines (DOPA-Lys-DOPA-Lys-DOPA) by a solid-phase peptide synthesis method. Then the active sequence of EGF and the adhesive peptide was conjugated using three glycine residues as a spacer. After purification with a high performance liquid chromatography (HPLC), formulations of the synthesized peptide were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass analysis (MALDI-TOF-MS). The binding affinity of EGF-DOPA to polystyrene (PSt) and titanium (Ti) surfaces was confirmed by using a quartz crystal microbalance with dissipation monitoring (QCM-D). The surface morphology and thickness of the substrates treated with EGF-DOPA was studied by atomic force microscopy (AFM), scanning electron microscopy (SEM) and ellipsometry. Rat kidney adherent fibroblast cell line, NRK49F, was cultured on the surfaces treated with EGF-DOPA. Cell growth and time course activation of cellular signal transduction was measured through WST-8 assay and phosphorylation analysis, respectively. Results and Discussion: MALDI-TOF-MS result showed that the expected 54-residue peptide (CPLSH-DGYCL-HDGVC-MYIEA-LDKYA-CNCW-GYIGE-RCQYR-DLKWW-E-GGG-DOPA-K-DOPA-K-DOPA, EGF-DOPA) was successfully synthesized. QCM measurement showed that the binding affinity of EGF-DOPA on both PSt and Ti surfaces was significantly higher than that of EGF. Concentration and pH dependent binding affinity of EGF-DOPA was confirmed. This result was also proved by thickness measurement using SEM and ellipsometry. The measured thicknesses of EGF-DOPA bound on the substrates displayed the same trend as those calculated from the QCM measurement. AFM results showed that aggregation of the EGF-DOPA happened at high pH condition. Substrates treated with EGF-DOPA significantly improved cell growth and this result can attributed to the long activation period of bound EGF-DOPA on the substrates. Conclusions: Mussel-inspired adhesive EGF derivative (EGF-DOPA) was constructed. The binding affinity of EGF-DOPA to both PSt and Ti surfaces was dramatically higher than that of EGF. The bound EGF-DOPA significantly promoted cell growth. This mussel-inspired dopamine modification provides an effective method for preparation of adhesive materials that may be useful for surface modification and the development of medical devices.