Effect of crosslinking chemistry of albumin/heparin multilayers on FGF-2 adsorption and endothelial cell behavior

Highlights: • Amine, carboxyl, and induced aldehyde groups of heparin were used for crosslinking of LbL film. • The carbodiimide-mediated cross-linking was the most efficient fixation method. • FGF-2 bound the most effectively to the glutaraldehyde-cross-linked LbL film. • Films cross-linked through carboxyl and aldehyde groups showed a slow FGF-2 release. • FGF-2-loaded LbL films stimulated the HUVECs growth better than the soluble FGF-2. - Abstract: The biomaterials that efficiently deliver growth factors represent an important challenge in cell-based tissue engineering. Layer-by-layer (LbL) thin films are attractive for incorporating controlled amounts of growth factors and releasing them over time. Herein, we investigated the effect of a method of cross-linking of albumin/heparin layer-by-layer (LbL) assembly ((Alb/Hep)₃) on the loading and release of basic fibroblast growth factor (FGF-2), and subsequent proliferation of human endothelial cells (HUVECs). The (Alb/Hep)₃ assemblies were cross-linked using glutaraldehyde, reductive amination or carbodiimide chemistries, and then biofunctionalized with FGF-2. The (Alb/Hep)₃ assemblies were characterized by the infrared multi-internal reflection spectroscopy, atomic force microscopy, ellipsometry, and surface plasmon resonance (SPR). The FGF-2 loading was quantified by the SPR in situ analysis. Our results showed that the (Alb/Hep)₃ cross-linking affected the amount of the bound heparin (from 150 to 315 ng/cm²), amount of FGF-2 loaded (from 75 to 125 ng/cm²), FGF-2 release (from 15 to 53% over 8 days), and consequently the HUVEC cell proliferation (from 50 to 80 × 10³ cells/cm² at day 5). All FGF-2 loaded assemblies stimulated the cell growth more than a soluble FGF-2 added into the cell media. In particular, the highest HUVECs proliferation was detected on the carbodiimide-cross-linked assembly. Overall, these biocompatible cross-linked assemblies can fine-tune the loading and release of growth factor providing a platform for cell-contacting applications.