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.

Polysaccharides in Ocular Drug Delivery

“…The half-lives were about ten times longer for the HA conjugates than for the unconjugated sFlt-1. Conjugation of sFlt-1 with HA did not change the pharmacological activity of this protein drug [170,171]. This study nicely demonstrated the potential use of polymer conjugation in intravitreal drug delivery.…”

“…Thus, the pharmacological effects of intravitreally administered drugs may be prolonged with increasing molecular size. Covalent conjugation to polymers can therefore be used to increase the apparent size of drug molecules [136], making the use of large polymer molecules a rational drug delivery strategy [169,170].…”

“…HA has been used to reduce clearance and increase the residence time of drugs in the vitreous humor [136]. Altiok et al [170,171] conjugated anti-VEGF drug (soluble VEGF receptor; sFlt-1) to HA (MW 300 kDa, 650 kDa, and 1 MDa) to provide sustained release and prolonged retention in the vitreous [170]. The half-lives were about ten times longer for the HA conjugates than for the unconjugated sFlt-1.…”

Biopolymer Molecular Weight Can Modulate the Wound Healing Efficacy of Multivalent Sonic Hedgehog–Hyaluronic Acid Conjugates

“…Previous in vitro studies using HyA protein conjugates have also shown that the R g,z is dependent on the HyA MW, and their transport properties through in vitro hydrogels is related to R g,z . 44 Thus, our findings suggest that it may be possible to optimize the mvShh parameters to present multiple Shh at a sufficiently high ligand spacing and also enhance its transport into the wound matrix. As a part of future studies, we will further investigate the mechanisms by which the HyA contributes to the pharmacokinetic properties of mvShh, as these studies will yield critical data to optimize their wound healing performance and advance their translation as a clinical therapy.…”

Expansion of bioorthogonal chemistries towards site-specific polymer–protein conjugation

“…HA possesses various biological activities, including cell migration, cell proliferation, antimicrobial, and wound healing. 225 221 In order to efficiently reduce the inflammation response induced by TNF-α, HAs were conjugated to anti-TNF-α antibodies through reactions between carboxyl acids of the HAs and amines of the antibodies.…”