Multivalent hyaluronic acid bioconjugates improve sFlt-1 activity in vitro.

Anti-VEGF drugs that are used in conjunction with laser ablation to treat patients with diabetic retinopathy suffer from short half-lives in the vitreous of the eye resulting in the need for frequent intravitreal injections. To improve the intravitreal half-life of anti-VEGF drugs, such as the VEGF decoy receptor sFlt-1, we developed multivalent bioconjugates of sFlt-1 grafted to linear hyaluronic acid (HyA) chains termed mvsFlt. Using size exclusion chromatography with multiangle light scattering (SEC-MALS), SDS-PAGE, and dynamic light scattering (DLS), we characterized the mvsFlt with a focus on the molecular weight contribution of protein and HyA components to the overall bioconjugate size. We found that mvsFlt activity was independent of HyA conjugation using a sandwich ELISA and in vitro angiogenesis assays including cell survival, migration and tube formation. Using an in vitro model of the vitreous with crosslinked HyA gels, we demonstrated that larger mvsFlt bioconjugates showed slowed release and mobility in these hydrogels compared to low molecular weight mvsFlt and unconjugated sFlt-1. Finally, we used an enzyme specific to sFlt-1 to show that conjugation to HyA shields sFlt-1 from protein degradation. Taken together, our findings suggest that mvsFlt bioconjugates retain VEGF binding affinity, shield sFlt-1 from enzymatic degradation, and their movement in hydrogel networks (in vitro model of the vitreous) is controlled by both bioconjugate size and hydrogel network mesh size. These results suggest that a strategy of multivalent conjugation could substantially improve drug residence time in the eye and potentially improve therapeutics for the treatment of diabetic retinopathy.

Enhanced survival and engraftment of transplanted stem cells using growth factor sequestering hydrogels.

We have generated a bioinspired tunable system of hyaluronic acid (HyA)-based hydrogels for Matrix-Assisted Cell Transplantation (MACT). With this material, we have independently evaluated matrix parameters such as adhesion peptide density, mechanical properties, and growth factor sequestering capacity, to engineer an environment that imbues donor cells with a milieu that promotes survival and engraftment with host tissues after transplantation. Using a versatile population of Sca-1(+)/CD45(-) cardiac progenitor cells (CPCs), we demonstrated that the addition of heparin in the HyA hydrogels was necessary to coordinate the presentation of TGFß1 and to support the trophic functions of the CPCs via endothelial cell differentiation and vascular like tubular network formation. Presentation of exogenous TGFß1 by binding with heparin improved differentiated CPC function by sequestering additional endogenously-produced angiogenic factors. Finally, we demonstrated that TGFß1 and heparin-containing HyA hydrogels can promote CPC survival when implanted subcutaneously into murine hind-limbs and encouraged their participation in the ensuing neovascular response, which included blood vessels that had anastomosed with the host's blood vessels.