Interaction of phosphorylcholine with fibronectin coatings: Surface characterization and biological performances

Highlights

• Fibronectin/phosphorylcholine coatings on plasma deposited fluorocarbon films were created.

• The effect of several coating techniques on the surface biological performances was evaluated.

• XPS, DWCA, immunostaining and ToF-SIMS (imaging and depth profiling) techniques were applied.

• Potential for cardiovascular applications was showed by endothelial cell and blood interactions.

Abstract

Coating medical devices with several bioactive molecules is an interesting approach to achieve specific biological targets upon the interaction of the biomaterial with the living environment. In this work, a fluorocarbon polymer (CF_x) was first deposited by plasma treatment on stainless steel (SS) substrate and thereafter, coatings containing fibronectin (FN) and phosphorylcholine (PRC) were created for cardiovascular applications. These two biomolecules were chosen to promote endothelialization and to avoid thrombus formation, respectively. Adsorption and grafting techniques were applied – and combined – to accomplish 4 different coatings containing both molecules. However, big challenge was found to characterize a small molecule (PRC: 184 g/mol) interacting with a protein (FN: 450 kD). For the first time XPS, dynamic water contact angle, immunostaining and ToF-SIMS (imaging and depth profiling) analyses were combined to accomplish the characterization of such a coating. The most encouraging biological performances were obtained for samples where FN was grafted to the CF_x film followed by the adsorption of PRC: proliferation of endothelial cells and hemocompatibility properties were observed. Promising coatings for cardiovascular applications were developed. The relevance of characterizing the coatings with high sensitive techniques and the further correlation with their biological performances were evidenced.

Introduction

The recent advances in materials science joined with an improved understanding of the function of biological systems have brought the ability to create synthetic materials with integrated biological activity, improving the performance of medical devices. In the specific case of cardiovascular stents, surface modification with bioactive molecules is desired for two main targets: inducing endothelialization and avoiding thrombus formation. In the present work, fibronectin (FN) and phosphorylcholine (PRC) were used to accomplish these two aims. FN is a glycoprotein of the extracellular matrix already studied to improve the adhesion and proliferation of endothelial cells [1], [2], [3], [4], [5]. PRC is a biomolecule found in the membranes of erythrocytes, well known for its non thrombogenic properties [6], [7], [8], [9]. The dual effect of different molecules has been already studied by different research groups: heparin/fibronectin [4], [10], collagen/heparin [11], chitosan/PRC [12], chitosan/heparin [13], PEG/antibodies [14], among others. In general, in these works only one coating technique is tested. However, in the present work four different coatings containing FN and PRC were created on a plasma-deposited fluorocarbon film (CF_x), by combining adsorption and grafting processes (Fig. 1). The CF_x film serves as a protection/barrier of the stainless steel substrate and at the same time, as a carrier for the biomolecules; its plasma deposition has been previously optimized by our team [15], [16]. Mainly due to the small size of PRC (184 gmol-1) compared to FN (450 kDa), a great defy emerged to characterize such coatings and hence to evaluate the effects of the coating technique in the biological performances of the surface. The common characterization techniques used regarding biomolecules and coatings, as well as some of their limitations are summarized in Table 1. The original approach of this paper is to combine different techniques: XPS, WCA, immunostaining and ToF-SIMS (both in static and depth profiling analyses), in order to understand the interaction of FN and PRC when adsorbed/grafted on the CF_x film. The surface characterization was then correlated to the biological performances of the surfaces: endothelial cell proliferation and haemolysis tests.