Directed evolution enables sequential, affinity-controlled delivery of angiogenic growth factors for vascular network formation

Introduction: Angiogenesis is a carefully coordinated process during which the temporally regulated presentation of many signaling proteins stimulates vascular network expansion. Disruptions in this sequence of protein presentation caused by injury and disease can impair tissue function. The objective of our work is to develop tunable biomaterials capable of phased therapeutic protein delivery to enhance angiogenesis. To achieve this objective, we have developed a “designer affinity” approach in which we control angiogenic protein delivery from hydrogels via specific, reversible affinity interactions with small, tethered protein domains called affibodies that we identified using directed evolution (Fig.1A). We will sequentially deliver three well-characterized angiogenic proteins from our biomaterial: vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and platelet-derived growth factor (PDGF). We expect this approach will recapitulate the natural, staggered presentation of proteins observed during angiogenesis and enhance vascular network formation.

Materials and

Methods: A yeast surface display library of 100 million affibodies underwent magnetic- and fluorescence-activated cell sorting to select for variants with affinity towards VEGF, FGF-2, or PDGF. The affinities of select affibodies were further tuned using Rosetta-based computational protein design. Yeast displaying protein-specific affibodies were incubated with 10-10,000nM of VEGF, FGF-2, or PDGF to determine dissociation constants (KD) between affibodies and growth factors using flow cytometry (Fig.1B). Affibodies were appended with an N-terminal adjacent hexa-histidine tag and cysteine, cloned into pet28b+ plasmids, and transformed into BL21(DE3) E. coli. Soluble affibodies were purified by immobilized metal affinity chromatography followed by size exclusion chromatography. Affibodies were conjugated into 5% (w/v) polyethylene glycol-maleimide (4-arm PEG-mal, 20kDa) hydrogels and crosslinked with dithiothreitol. Hydrogels were loaded with growth factors at a 500:1 affibody:growth factor ratio. Protein release at 37 C over 7 days was quantified using enzyme-linked immunosorbent assays. Microvascular fragments (MVFs), containing endothelial cells, pericytes, and stromal cells, were harvested from 6-month-old Lewis rat epididymal fat pads and seeded in 0.3% (w/v) collagen hydrogels at 20,000 fragments/mL. MVFs received 50nM of VEGF, FGF-2, and/or PDGF at day 3 (“codelivery”) or 50nM VEGF, FGF-2, and/or PDGF at day 3, 4, and 5 (“sequential”). After 7 days, MVFs were fixed, stained with lectin, and imaged. Each confocal z-stack was 3D median filtered and deconvolved before volumetric segmentation and morphological assessment were performed in Amira Software to measure branching and total network length. Vascular network metrics were normalized to MVFs without growth factors (media only).

Results, Conclusions, and Discussions: Three VEGF-, three PDGF-, and three FGF-2-specific affibodies were discovered using a combination of yeast surface display and computational design, with dissociation constants spanning four orders magnitude (3.07nM-6470nM) and broadly characterized as high, medium, and low affinity affibodies (Fig.1B). Affinity characterization of the two remaining PDGF affibodies is currently underway. These affibodies are also highly specific, displaying affinity only for the target protein. When solubly expressed in E. coli, all affibodies exhibited the expected alpha-helical structure as measured via circular dichroism. Affibody-conjugated PEG-mal hydrogels initially encapsulated >85% of loaded growth factors. Less protein was released from hydrogels containing affibodies with higher affinities for their target proteins, demonstrating an inverse relationship between affibody affinity and protein release and indicating the ability of affibodies to tune protein release (Fig.1C). Sequential delivery of VEGF followed by FGF-2 significantly increased MVF network length compared to simultaneous codelivery of VEGF and FGF-2 (Fig.1D), suggesting a role of temporal regulation of growth factor signaling in vascular network expansion. Fig.1E depicts the visual difference in MVF vascular network observed via confocal microscopy in the absence or presence of sequentially delivered angiogenic growth factors.

In conclusion, we have identified a library of novel affibodies with high specificity and a wide range of affinities for VEGF, FGF-2, and PDGF. We have demonstrated that these affibodies can tune VEGF, FGF-2, and PDGF release from hydrogels and that sequential protein delivery enhances vascular network formation compared to simultaneous codelivery. In the future, we will investigate additional sequences and timing of protein delivery before tuning our hydrogel platform to provide this delivery sequence. Overall, our work presents an innovative, highly tunable platform with the potential for enhancing angiogenesis.

Acknowledgements (Optional): This work was supported by an Oregon Health & Science University Medical Research Foundation New Investigator grant, National Institutes of Health (NIH) Maximizing Investigators' Research Award (R35GM147507), and the National Science Foundation (NSF) Molecular Probes and Sensors for Complex Environments (MoPSCE) Training Program.