Phased affinity-controlled delivery of vascular endothelial growth factor, fibroblast growth factor-2, and platelet derived growth factor enhances in vitro angiogenesis.

Angiogenesis, the growth of vasculature from existing blood vessels, requires the coordinated secretion of multiple angiogenic growth factors that each stimulate the cellular recruitment, patterning, and morphogenesis inherent to vascular network formation. Among these secreted factors, vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and platelet derived growth factor (PDGF) amplify key stages of angiogenesis. Disruptions in their secretion have been implicated in poor vascular network formation. Current methods for exploring variations in the phased presentation of multiple different proteins are limited, which has restricted our ability to explore the effect of growth factor timing on angiogenesis. To address this knowledge gap, we developed affibodies, which are alpha-helical binding proteins, to phase the release of VEGF-165, FGF-2, and PDGF-BB from a single drug delivery vehicle via specific protein-affibody affinity interactions. We used yeast surface display to engineer three VEGF-, three FGF-2-, and two PDGF-specific affibodies with different affinities for their target proteins. We demonstrated that the cumulative release of VEGF and FGF-2 are inversely correlated with the strength of the protein-affibody affinity interaction and that hydrogels containing multiple protein-specific affibodies can independently control the release of VEGF, FGF-2, and PDGF, largely in accordance with the strength of the affinity interactions. Using a rat-derived intact microvascular fragment model of in vitro angiogenesis, we revealed that sequential delivery of soluble VEGF, followed by FGF-2, and then PDGF enhances vascular network formation and branching. We then designed an affibody-conjugated hydrogel to mimic this sequence of protein delivery, resulting in increased vascular branching and network length compared to all other hydrogel compositions and the sequential delivery of soluble growth factors. This work establishes a new platform for modulating the timing of growth factor delivery, enabling the exploration of how temporal variations in protein secretion impact regeneration and development.