A biomimetic peptide fluorosurfactant polymer for endothelialization of ePTFE with limited platelet adhesionIntroductionMaterials and methodsPeptide fluorosurfactant polymer synthesis and characterizationPeptide synthesis, cyclization, and purificationSynthesis of peptide fluorosurfactant polymerUV spectroscopySolid-phase integrin binding competitionPlatelet aggregometrySurface preparation and modificationCell culturePeptide and integrin specificityAdhesion and growth studiesShear stability testingHemostatic function studiesPlatelet adhesion to surfacesStatistical analysisResults and discussionSolid-phase alphaIIbbeta3 integrin binding competition and platelet aggregometryPeptide fluorosurfactant polymer synthesis and characterizationPeptide and integrin specificity of EC attachmentEC attachment and growthEC shear stabilityEC hemostatic functionPlatelet adhesionConclusionsAcknowledgmentsReferencesBiomaterials 28 (2007) 3537–3548A biomimetic peptide fluorosurfactant polymer for endothelialization ofePTFE with limited platelet adhesionCoby C. Larsena, Faina Kligmanb, Chad Tanga,Kandice Kottke-Marchanta,b, Roger E. Marchanta,aDepartment of Biomedical Engineering, Case Western Reserve University, 10900 Euclid Ave., Wickenden 309, Cleveland, OH 44106, USAbDepartment of Clinical Pathology, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195, USAReceived 24 January 2007; accepted 3 April 2007Available online 4 May 2007AbstractEndothelialization of expanded polytetrafluoroethylene (ePTFE) has the potential to improve long-term patency for small-diametervascular 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 tounderlying ePTFE. The EC-selective peptide (primary sequence: Cys–Arg–Arg–Glu–Thr–Ala–Trp–Ala–Cys, CRRETAWAC) hasdemonstrated high binding affinity for the a5b1integrin found on ECs. Here, we demonstrate low affinity of CRRETAWAC for plateletsand platelet integrins, thus providing it with EC-selectivity. This EC-selectivity could potentially facilitate rapid in vivo endothelializationand healing without thrombosis for small-diameter ePTFE vascular grafts.r 2007 Elsevier Ltd. All rights reserved.Keywords: Endothelial cells; ePTFE vascular grafts; Surface modification; Platelet adhesion1. IntroductionThere is a pressing clinical need for suitable small-diameter vascular prostheses to bypass diseased coronaryand peripheral arteries. Mater ials, such as expandedpolytetrafluoroethylene (ePTFE), that are successful aslarge-diameter vascular prostheses have proven inadequatewhen used in small-diameter applications because ofthrombosis and occlusion [1,2]. The ideal blood interfaceis a confluent layer of healthy endothelial cells (ECs). Pre-implantation endothelialization of ePTFE has demon-strated limited clinical improvement in long-term patencyfor small-diameter grafts [3,4]. The challenge of tissueengineering the ideal blood interface is that the samematrix proteins (e.g. fibronectin—FN) or FN-derivedpeptides that bind ECs also will bind platelets and initiatethrombosis. We have previously reported a peptidefluorosurfactant polymer (FSP) modification of ePTFEthat facilitates EC adhesion, growth and function [5].However, the RGD-containing peptide sequence used forcell binding on our previous construct has roughlyequivalent affinity for EC and platelet integrins [6–8].Inthis study, we hypothesized that platelet bind ing could bereduced while maintaining EC adhesion, growth, shearstability, and hemostatic function if an alternative, ECintegrin-selective peptide was employed in our FSPbiomimetic construct.The cyclic peptide sequence Cys–Arg–Arg–Glu–Thr–Ala–Trp–Ala–Cys (CRRETAWAC; Fig. 1) has beenshown to bind with high specificity and affinity to thea5b1integrin (IC50for a5b1integrin binding is 0.01 mM,but 41000 mM for avb3integrin) [9]; this has beenattributed to RRE motif interaction with the b1subunit(similar to RGD interaction) [10] and Trp interaction witha Trp residue in the a5subunit [11–13]. High affinityARTICLE IN PRESSwww.elsevier.com/locate/biomaterials0142-9612/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.biomaterials.2007.04.026Corresponding author. Tel.: +1 216 368 3005; fax: +1 216 368 4969.E-mail address: [email protected] (R.E. Marchant).binding of CRRETAWAC to a5b1integrin confers EC-selectivity due to the relative prominence of a5b1integrinon ECs compared with platelets [14].In this paper, we examine the binding affinity of cyclicCRRETAWAC for platelet receptors, specifically theaIIbb3integrin. The synthesis and characterization ofCRRETAWAC FSP (Fig. 1) is detailed. We also investi-gate EC adhesion, growth, shear stability, and function onCRRETAWAC FSP-modified fluorocarbon substr ates.Finally, we demonstrate limited platelet adhesion onCRRETAWAC FSP, establishing its potential to promoterapid endothelialization without thrombosis for small-diameter vascular grafts.2. Materials and methods2.1. Peptide fluorosurfactant polymer synthesis andcharacterization2.1.1. Peptide synthesis, cyclization, and purificationThe cell adhesive peptide was synthesized using an Applied Biosystems(model 433A) solid-phase peptide synthesizer, utilizing 9-fluorenylmethoxy-carbonyl (Fmoc) methodology, common solvents, packing resin and cappedamino acids. This peptide is a 13 amino acid molecule having the followingsequence: CRRETAWACSSSG (Fig. 1, or negative control scrambledpeptide CATAERWRCSSSG). Poly(vinyl amine) (PVAm, Mn ¼ 11,000)was synthesized as described [15] with molecular weight characterized byGPC using a cationic column and a laser scattering detector [16].Thehydrophilic SSSG spacer present at the N-terminus of the peptide wasincluded to allow for elevation of the functional CRRETAWAC peptidesequence from the PVAm backbone. Formation of disulfide bonds betweensulfhydryl group of cysteine residues was achieved by a solution oxidationmethod [17]. The linear peptide (150 mg), cleaved and deprotected,