A biomimetic peptide fluorosurfactant polymer for endothelialization of ePTFE with limited platelet adhesion

Abstract

Endothelialization of expanded polytetrafluoroethylene (ePTFE) has the potential to improve long-term patency for small-diameter vascular grafts. Successful endothelialization requires ePTFE surface modification to permit cell attachment to this otherwise non-adhesive substrate. We report here on a peptide fluorosurfactant polymer (FSP) biomimetic construct that promotes endothelial cell (EC)-selective attachment, growth, shear stability, and function on ePTFE. The peptide FSP consists of a flexible poly(vinyl amine) backbone with EC-selective peptide ligands for specific cell adhesion and pendant fluorocarbon branches for stable anchorage to underlying ePTFE. The EC-selective peptide (primary sequence: Cys-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys, CRRETAWAC) has demonstrated high binding affinity for the alpha(5)beta(1) integrin found on ECs. Here, we demonstrate low affinity of CRRETAWAC for platelets and platelet integrins, thus providing it with EC-selectivity. This EC-selectivity could potentially facilitate rapid in vivo endothelialization and healing without thrombosis for small-diameter ePTFE vascular grafts.

Figure 1

Reaction scheme for synthesis of fluorosurfactant polymer with CRRETAWAC peptide. The RRE motif is believed to interact with β₁ integrin subunit [10]; the Trp residue is believed to interact with Trp¹⁵⁷ within the α₅ integrin subunit [11]. Peptide FSP consists of poly(vinyl amine) backbone with free amines (x=7), glutaraldehyde linked cyclic GSSSCRRETAWAC cell-binding peptide (y=2), and substrate adsorbing perfluorocarbon pendant branches (z=1).

Figure 2

Platelet interaction with CRRETAWAC. A) Inhibition of fibrinogen binding to immobilized α_IIbβ₃ platelet integrin by GRGDSP () or CRRETAWAC () with nonlinear logistic regression fits. Higher IC₅₀ for CRRETAWAC indicates lower affinity for α_IIbβ₃ integrin. B) Inhibition of platelet aggregation by GRGDSP () or CRRETAWAC () with nonlinear logistic regression fits. Higher IC₅₀ for CRRETAWAC indicates lower affinity for platelet receptors involved in aggregation.

Figure 3

Stability of CRRETAWAC FSP on PTFE in aqueous conditions. Change in relative nitrogen content (%N ) after exposure to static aqueous conditions for 4 and 20 weeks. Bars represent standard deviation for 3 measurements for each data point.

Figure 4

Endothelial cell (EC) specificity of attachment to CRRETAWAC fluorosurfactant polymer (FSP). A) CRRETAWAC dependence of EC attachment to CRRETAWAC FSP. ECs pre-incubated with soluble CRRETAWAC showed reduced attachment to CRRETAWAC FSP compared with cells pre-incubated with CATAERWRC or no peptide. * Significantly different (p<0.05) cell attachment compared with no peptide pre-treatment. B) α₅β₁ integrin specificity of EC attachment to CRRETAWAC FSP. Cells pre-incubated with α₅β₁ integrin blocking antibody (Ab) demonstrated significantly reduced attachment to CRRETAWAC FSP, indicating that the α₅β₁ integrin is principally responsible for cell attachment. * Significantly different (p<0.05) cell attachment compared with no Ab pre-treatment for same surface type. # Significantly different (p<0.05) cell attachment compared with same Ab pre-treatment for FN surface. Bars represent standard deviation for at least 3 replicates per data point.

Figure 5

Endothelial cell (EC) attachment and growth on unmodified ePTFE, fibronectin (FN), and CRRETAWAC fluorosurfactant polymer (FSP) modified ePTFE and FSAM. Cell attachment and growth on CRRETAWAC FSP was higher than on FN and substantially greater than on unmodified ePTFE. ^# Significantly different (p<0.05) cell population compared with FN and ePTFE surfaces at the same time point. * Significantly different (p<0.05) cell population compared with FN and CRRETAWAC FSP surfaces at the same time point. Bars represent standard deviation for at least 3 replicates per data point.

Figure 6

Endothelial cell (EC) shear stability and morphology on CRRETAWAC fluorosurfactant polymer (FSP). A) EC shear stability on FN (△), CRRETAWAC FSP (), and RGD FSP (). ECs remained stably adherent to CRRETAWAC FSP after 4 h of 47.8 dynes/cm² applied shear stress. * Significant difference in cell population (p<0.05) compared with 9.6 dynes/cm² shear stress for same surface. B) Alignment of ECs on FN, CRRETAWAC FSP, and RGD FSP after 4 h of applied shear stress. ECs reoriented and aligned with shear upon application of minimal shear stress for all surfaces. * Significantly different (p<0.05) % of aligned cells compared with 9.6 and 38.2 dynes/cm² shear stress for the same surface type. Bars represent standard deviation for 4 replicates per data point. C) Actin cytoskeleton staining for ECs on FN displaying alignment with shear after 4 h at 28.7 dynes/cm². Scale bars for images are 100 μm. Arrows indicates direction of applied shear stress. D) Actin cytoskeleton staining for ECs on CRRETAWAC FSP displaying alignment with shear after 4 h at 28.7 dynes/cm². E) Actin cytoskeleton staining for ECs on RGD FSP displaying alignment with shear after 4 h at 28.7 dynes/cm².

Figure 7

Endothelial cell (EC) hemostatic function on fibronectin (FN) and peptide fluorosurfactant polymer (FSP) surfaces. A) Prostacyclin production by ECs on FN, CRRETAWAC FSP, and RGD FSP as measured by a surrogate breakdown product, 6-keto prostaglandin F_1α (PGF_1α). Cellular production values (pg/cell/h) were normalized to the production rate from ECs on FN for a particular experimental run. * Significantly different (p<0.05) normalized production compared with ECs on FN and RGD FSP surfaces. Bars represent standard deviation for at least 6 replicates per data point. B) Equivalent tissue plasminogen activator (tPA) production by ECs on FN, CRRETAWAC FSP, and RGD FSP. Cellular production values (ng/cell/h) were normalized to the production rate from ECs on FN for a particular experimental run. Bars represent standard deviation for at least 6 replicates per data point. C) Uptake of fluorescently labeled acetylated low density lipoprotein (red) by ECs (nuclei are blue) adherent to CRRETAWAC FSP modified FSAM. Scale bar is 100 μm.

Figure 8

Platelet interaction with CRRETAWAC fluorosurfactant polymer (FSP) surface. A) Platelet surface coverage for FN, CRRETAWAC FSP, and RGD FSP. * Significantly different (p<0.05) platelet surface coverage for CRRETWAC FSP compared with FN and RGD FSP. Bars represent standard deviation for at least 3 replicates per data point. B) FITC-anti CD41a labeled platelets adherent to CRRETAWAC FSP modified FSAM. Scale bars for all images are 30 μm. C) FITC-anti CD41a labeled platelets adherent to fibronectin (FN) coated glass. D) FITC-anti CD41a labeled platelets adherent to RGD FSP modified FSAM.