Thiol-ene conjugation of VEGF peptide to electrospun scaffolds as potential application for angiogenesis

Abstract

Vascular endothelial growth factor (VEGF) plays a vital role in promoting attachment and proliferation of endothelial cells, and induces angiogenesis. In recent years, much research has been conducted on functionalization of tissue engineering scaffolds with VEGF or VEGF-mimetic peptide to promote angiogenesis. However, most chemical reactions are nonspecific and require organic solvents, which can compromise control over functionalization and alter peptide/protein activity. An attractive alternative is the fabrication of functionalizable electrospun fibers, which can overcome these hurdles. In this study, we used thiol-ene chemistry for the conjugation of a VEGF-mimetic peptide to the surface of poly (ε-caprolactone) (PCL) fibrous scaffolds with varying amounts of a functional PCL-diacrylate (PCL-DA) polymer. 30% PCL-DA was selected due to homogeneous fiber morphology. A VEGF-mimetic peptide was then immobilized on PCL-DA fibrous scaffolds by a light-initiated thiol-ene reaction. 7-Mercapto-4-methylcoumarin, RGD-FITC peptide and VEGF-TAMRA mimetic peptide were used to validate the thiol-ene reaction with fibrous scaffolds. Tensile strength and elastic modulus of 30% PCL-DA fibrous scaffolds were significantly increased after the reaction. Conjugation of 30% PCL-DA fibrous scaffolds with VEGF peptide increased the surface water wettability of the scaffolds. Patterned structures could be obtained after using a photomask on the fibrous film. Moreover, in vitro studies indicated that scaffolds functionalized with the VEGF-mimetic peptide were able to induce phosphorylation of VEGF receptor and enhanced HUVECs survival, proliferation and adhesion. A chick chorioallantoic membrane (CAM) assay further indicated that the VEGF peptide functionalized scaffolds are able to promote angiogenesis in vivo. These results show that scaffold functionalization can be controlled via a simple polymer mixing approach, and that the functionalized VEGF peptide-scaffolds have potential for vascular tissue regeneration.

Keywords: Electrospun; Fibrous scaffolds; Thiol-ene reaction; VEGF peptide.

Graphical abstract

Fig. 1

Micro- or nano-structure of electrospun fibrous scaffolds; (a and d) 10% PCL-DA, (b and e) 30% PCL-DA, and (c and f) 50% PCL-DA at low (top) and high (bottom) magnifications. Scale bars are 100 μm (a–c) and 10 μm (b–d).

Fig. 2

(a) The effect of reaction time on the fluorescence increases of 30% PCL-DA fibrous scaffolds. The control group is the reaction group without UV treatment. Excitation wavelength: 335 nm. (b) Quantification of 7-Mercapto-4-methylcoumarin reacted with per 10, 30 and 50% of the PCL-DA electrospun scaffolds, respectively. (*P ≤ 0.05, ***P ≤ 0.001; n = 3).

Fig. 3

(a) Photopatterning steps of 30%PCL-DA fibrous scaffolds with 7-Mercapto-4-methylcoumarin. (b) Images of mask and photopatterned fibrous scaffolds and (c) different mask and photopatterned fibrous scaffolds.

Fig. 4

(a) VEGF-TAMRA mimic peptide immobilizing on different PCL-DA electrospun scaffolds. Up: 10% PCL-DA scaffolds; Down: 50% PCL-DA scaffolds. (b) Quantification of VEGF-TAMRA concentration in the different electrospun scaffolds. Scale bars are 100 μm. (**P ≤ 0.01, ***P ≤ 0.001; n = 3).

Fig. 5

Representative tensile stress–strain curves (a) and elastic modulus (b) of 30% PCL-DA and 30% PCL-DA/VEGF pep fibrous scaffolds. (*P ≤ 0.05; n = 3).

Fig. 6

Effect of VEGF peptide immobilizing on HUVECs survival under nutrient starvation conditions. Viability (a) and DNA content (b) of HUVECs cultured for 5 days on Matrigel coated 30% PCL-DA and 30% PCL-DA/VEGF pep fibrous scaffolds. (**P ≤ 0.01; n = 3).

Fig. 7

ELISA analysis of (a) phospho-VEGFR1 and (b) phospho-VEGFR2 production of HUVECs on uncoated 30%PCL-DA and 30% PCL-DA/VEGF pep fibrous scaffolds after 2 h and 4 h “30% PCL-DA + VEGF” represent HUVECs cultured on 30% PCL-DA fibrous scaffolds with adding VEGF in medium. (*P ≤ 0.05; n = 3).

Fig. 8

The viability (a) and proliferation (b) of HUVECs on uncoated 30% PCL-DA and 30% PCL-DA/VEGF pep fibrous scaffolds in EGM medium without VEGF during 5 days of culture. (*P ≤ 0.05; n = 3).

Fig. 9

Processed images of uncoated (a) 30% PCL-DA and (b) 30% PCL-DA/VEGF pep fibrous scaffolds implanted on the CAM after 4 days. The quantification of (c) vessel area in the images and (d) number of vessels around implanted scaffolds. (**P ≤ 0.01; n = 10).