Immobilization of glycoproteins, such as VEGF, on biodegradable substrates

Abstract

Attachment of growth factors to biodegradable polymers, such as poly(lactide-co-glycolide) (PLGA), may enhance and/or accelerate integration of tissue engineering scaffolds. Although proteins are commonly bound via abundant amino groups, a more selective approach may increase bioactivity of immobilized molecules. In this research, exposed carboxyl groups on acid-terminated PLGA were modified with dihydrazide spacer molecules. The number of hydrazide groups available for subsequent attachment of protein was dependent on dihydrazide length, with shorter molecules present at significantly greater surface densities. The potent angiogenic glycoprotein vascular endothelial growth factor (VEGF) was oxidized with periodate and the aldehyde moieties allowed to react with the hydrazide-derivatized PLGA. Derivatization initially affected the amount of protein bound to the surfaces, but differences were substantially reduced following overnight incubation in saline. More importantly, use of shorter dihydrazide spacers significantly enhanced accessibility of immobilized VEGF for binding neutralizing antibody and soluble VEGF receptor. Furthermore, immobilized growth factor enhanced endothelial cell proliferation, with surfaces having the shortest and longest spacers stimulating greater effects. The present work has not only demonstrated an alternative approach to immobilizing growth factors on biodegradable materials, but the scheme can be used to alter the amount of protein bound as well as its availability for subsequent biointeractions.

Figure 1

Structure of dihydrazide spacer molecules having increasing length.

Figure 2

Idealized scheme for dihydrazide attachment to PLGA.

Figure 3

Idealized scheme for VEGF attachment to dihydrazide-modified PLGA.

Figure 4

Surface density of hydrazide groups available on PLGA following derivatization with dihydrazide spacers of increasing length.

Figure 5

Amount of VEGF bound to hydrazide-derivatized PLGA and directly on carbodiimide-activated PLGA surfaces initially and following overnight incubation in PBS.

Figure 6

Accessibility of VEGF immobilized on hydrazide-derivatized PLGA and directly on carbodiimide-activated PLGA surfaces. (a) Binding of neutralizing antibody before and after overnight incubation in PBS. (b) Binding of sVEGF R2/Fc chimera following overnight incubation in PBS.

Figure 6

Accessibility of VEGF immobilized on hydrazide-derivatized PLGA and directly on carbodiimide-activated PLGA surfaces. (a) Binding of neutralizing antibody before and after overnight incubation in PBS. (b) Binding of sVEGF R2/Fc chimera following overnight incubation in PBS.

Figure 7

Proliferation of endothelial cells seeded on VEGF bound to hydrazide-derivatized PLGA and directly on carbodiimide-activated PLGA surfaces following overnight incubation in PBS. For comparison, cells were also incubated with 10 ng ml⁻¹ soluble VEGF.