The effect of RGD fluorosurfactant polymer modification of ePTFE on endothelial cell adhesion, growth, and function

Abstract

We have synthesized and characterized a novel peptide fluorosurfactant polymer (PFSP) modification that facilitates the adhesion and growth of endothelial cells on expanded polytetrafluoroetheylene (ePTFE) vascular graft material. This PFSP consists of a poly(vinyl amine) (PVAm) backbone with integrin binding Arg-Gly-Asp (RGD) peptides and perfluorocarbon pendant branches for adsorption and stable adhesion to underlying ePTFE. Aqueous PFSP solution was used to modify the surface of fluorocarbon substrates. Following subconfluent seeding, endothelial cell (EC) adhesion and growth on PFSP was assessed by determining cell population at different time points. Spectroscopic results indicated successful synthesis of PFSP. PFSP modification of ePTFE reduced the receding water contact angle measurement from 120 degrees to 6 degrees , indicating successful surface modification. Quantification of cell population demonstrated reduced EC attachment efficiency but increased growth rate on RGD PFSP compared with fibronectin (FN). Actin staining revealed a well-developed cytoskeleton for ECs on RGD PFSP indicative of stable adhesion. Uptake of acetylated low-density lipoprotein and positive staining for VE-Cadherin confirm EC phenotype for adherent cells. Production of prostacyclin, a potent antiplatelet agent, was equivalent between ECs on FN and RGD PFSP surfaces. Our results indicate successful synthesis and surface modification with PFSP; this is a simple, quantitative, and effective approach to modifying ePTFE to encourage endothelial cell attachment, growth, and function.

Figure 1

Synthetic strategy for peptide fluorosurfactant polymer.

Figure 2

IR spectra of PVAm-RGD-peptide conjugate (1) and PVAm with attached RGD peptide and perfluorocarbon (2)

Figure 3

Plot of surface tension versus logarithm of concentration for peptide (•), peptide-aldehyde (■), and peptide fluorosurfactant polymer (▲).

Figure 4

Aqueous desorption stability. Change in a.) relative nitrogen content or b.) advancing (θ_a-filled symbols) and receding (θ_r-unfilled symbols) water contact angle on RGD peptide fluorosurfactant polymer on PTFE (θ_a ■, θ_r □, %N ■) after exposure to static aqueous conditions for various time lengths.

Figure 5

EC adhesion and growth on RGE peptide fluorosurfactant polymer (PFSP), RGD PFSP, and fibronectin (FN) surfaces. * indicates difference (p<0.05) in EC population from RGD PFSP surface at same time point. # indicates difference (p<0.05) in EC population from identical surface at 20 h post-seeding. Error bars are 95% confidence intervals from 7 FN samples and 10 RGD PFSP surfaces at each time point.

Figure 6

EC morphology and surface coverage visualized with phalloidin (a & d), phenotype and function indicated by DiI AcLDL uptake (b & e) and VE-Cadherin staining (c & f; arrows) on RGD PFSP (a- FSAM substrate, b and c- ePTFE substrate) and FN (d–f). Scale bars are 100 μm.

Figure 7

Production of prostacyclin by ECs on FN and RGD PFSP measured with an enzyme immunoassay for 6-keto PGF_1α, a hydrolysis product of prostacyclin. Bars represent standard deviation of 3 samples.

Figure 8

Effect of soluble GRGESP or GRGDSP on endothelial cell (EC) attachment to RGD peptide fluorosurfactant polymer (PFSP) or fibronectin (FN) surfaces. * indicates significantly different (p<0.001) mean CellTiter assay absorbance compared with ECs incubated with 1 mM GRGDSP and seeded on RGD PFSP surface. Data are presented ± standard deviations from n = 4.