title: Multivalent hyaluronic acid bioconjugates improve sFlt-1 activity in vitro.
abstract: 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.
authors.affiliation: Department of Bioengineering, University of California at Berkeley, CA 94720, USA.
authors.forename: Eda I
authors.initials: EI
authors.lastname: Altiok
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California at Berkeley, CA 94720, USA.
authors.forename: Jorge L
authors.initials: JL
authors.lastname: Santiago-Ortiz
authors.affiliation: Department of Materials Science and Engineering, University of California at Berkeley, CA 94720, USA.
authors.forename: Felicia L
authors.initials: FL
authors.lastname: Svedlund
authors.affiliation: Department of Bioengineering, University of California at Berkeley, CA 94720, USA.
authors.forename: Aline
authors.initials: A
authors.lastname: Zbinden
authors.affiliation: Department of Bioengineering, University of California at Berkeley, CA 94720, USA.
authors.forename: Amit K
authors.initials: AK
authors.lastname: Jha
authors.affiliation: Department of Bioengineering, University of California at Berkeley, CA 94720, USA.
authors.forename: Deepika
authors.initials: D
authors.lastname: Bhatnagar
authors.affiliation: Department of Bioengineering, University of California at Berkeley, CA 94720, USA; Department of Materials Science and Engineering, University of California at Berkeley, CA 94720, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Loskill
authors.affiliation: Department of Bioengineering, University of California at Berkeley, CA 94720, USA.
authors.forename: Wesley M
authors.initials: WM
authors.lastname: Jackson
authors.affiliation: Department of Bioengineering, University of California at Berkeley, CA 94720, USA; Department of Chemical and Biomolecular Engineering, University of California at Berkeley, CA 94720, USA.
authors.forename: David V
authors.initials: DV
authors.lastname: Schaffer
authors.affiliation: Department of Bioengineering, University of California at Berkeley, CA 94720, USA; Department of Materials Science and Engineering, University of California at Berkeley, CA 94720, USA. Electronic address: kehealy@berkeley.edu.
authors.forename: Kevin E
authors.initials: KE
authors.lastname: Healy
mesh_terms: D001672:Biocompatible Materials / Q000737:chemistry*; D002465:Cell Movement; D002850:Chromatography, Gel; D000067493:Dynamic Light Scattering; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry*; D020783:Matrix Metalloproteinase 7 / Q000378:metabolism; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000378:metabolism*
chemical_list: D001672:Biocompatible Materials; D006820:Hyaluronic Acid; D040281:Vascular Endothelial Growth Factor Receptor-1; D020783:Matrix Metalloproteinase 7
keywords: Angiogenesis; Anti-VEGF drug; Drug delivery; Hyaluronic acid