Elsevier

European Polymer Journal

Volume 58, September 2014, Pages 11-22
European Polymer Journal

Poly(ethylene oxide) brushes prepared by the “grafting to” method as a platform for the assessment of cell receptor–ligand binding

https://doi.org/10.1016/j.eurpolymj.2014.06.004Get rights and content

Highlights

  • PEO/PGMA layers with PEO thickness up to 30 nm are stable in buffered saline for 8 days.

  • PEO/PGMA layers degrade by detachment in the form of a continuous film.

  • CuAAC coupling of a peptide ligand allows using the full capacity of the PEO layer.

  • Cell adhesion is promoted by the RGD peptide at concentrations of 101 pmol/cm2.

  • Optimal PEO thickness for the ligand–cell receptor assay ranges from 10 nm to 20 nm.

Abstract

Poly(ethylene oxide) (PEO) with terminal alkyne and amino groups was grafted to a poly(glycidyl methacrylate) (PGMA) anchoring layer and the PEO/PGMA coatings were investigated as a non-fouling platform for the assessment of ligand–cell receptor interactions. The PEO/PGMA coatings deposited on Si/SiO2 substrate were stable in phosphate buffered saline over a period of 8 days if the thickness of the PEO was less than 30 nm. The stability of the coating could be enhanced by an additional layer of 3-mercaptopropyltrimethoxysilane between the substrate and the PGMA layer. The grafted layers were shown to efficiently suppress nonspecific protein adsorption and cell adhesion. Based on the theoretical ligand-binding capacity, protein adsorption and stability data, the optimum thickness range of the PEO layer is 10–20 nm. The binding of an arginine–glycine–aspartic acid (RGD) ligand using azide–alkyne click chemistry demonstrated that the ligand surface density is controllable in the range from 100 pmol/cm2 up to the capacity of the grafted layer of 102 pmol/cm2 by varying the ligand concentration in the reaction mixture. Calf pulmonary artery endothelial cells adhered to and spread on ligand modified layers proportionally to the ligand surface density, thus demonstrating the applicability of these “grafted to” PEO brushes as a platform for cell receptor–ligand engagement studies.

Introduction

Synthetic peptide ligands derived from adhesion sites of extracellular matrix proteins are effective tools in guiding cell behavior on biomaterial surfaces [1], [2]. Evaluating the interactions between cell membrane receptors and synthetic ligands is therefore a crucial issue in the development of advanced biomaterials for regenerative medicine and tissue engineering [3], [4], [5]. In order to elicit specific and controlled binding between ligands and cells, the ligands have to be presented against a background resistant to the adsorption of proteins from culture media, which would otherwise compromise the specificity of the assay [6], [7].

Cell–ligand interactions are typically assessed on model planar substrates coated with well-defined self-assembled monolayers of functional thiols [8], [9] or silanes [10]. More stable, versatile and substrate-independent coatings are prepared from low-fouling hydrophilic polymers [7]. These coatings have a great potential for use not only as testing surfaces but also as functional coatings in biomedical devices. Various structures of these polymer-based coatings have been developed, including interpenetrating networks [11], multicomponent cross-linked layers [12], polymer star layers [13], polymer brushes [14], [15], [16] and plasma polymer layers [17]. Among these, polymer brushes meet the requirements of cell–ligand interaction assays, namely reproducibility, predetermined ligand-binding capacity and good resistance to a non-specific protein adsorption. Polymer brushes are mostly prepared by a “grafting from” approach via surface initiated controlled radical polymerization [18], which provides precise control over the brush thickness and composition. Alternatively, they can be prepared by a “grafting to” approach, i.e., by attaching end-functionalized ready-made polymers to a chemically activated surface. The brush state can be reached if the grafting is performed either from a polymer melt or from a polymer film plasticized by solvent vapors (solvent-assisted grafting) [19], [20], i.e., under conditions when the excluded volume interactions limiting the grafting density are efficiently screened. In comparison with “grafting from”, the “grafting to” method is experimentally very straightforward. It avoids the polymerization step in a closed reactor and so it is a convenient way to prepare larger batches of samples.

Both grafting methods need surface functional groups that serve either as initiating sites or as coupling sites. To increase the independence from the substrate and the efficiency of the grafting, a macromolecular anchoring layer bearing reactive groups both for grafting and for anchoring to the substrate has been used [19], [21], [22]. A monolayer of the anchoring polymer bound to the surface provides reactive groups for grafting localized in tails and loops between the anchoring sites. This quasi three-dimensional layer has much higher grafting capacity and efficiency than native functional groups of the material surface or those introduced through a surface assembly of low-molecular weight modifiers, e.g. silanes.

The concept of the “grafting to” method, in combination with a macromolecular anchoring layer for preparing high-density polymer brushes, has been developed largely by the group of Luzinov [19], [23], [24]. They suggested the use of epoxy group-containing polymers such as poly(glycidyl methacrylate) (PGMA) for the anchoring layer [25]. Epoxy groups are able to react not only with typical nucleophiles such as amino and thiol groups but also with less reactive surface hydroxyl groups typically present on many inorganic materials, such as metals, semimetals and silicates [26], [27].

Poly(ethylene oxide) (PEO), which is a well-established component of low-fouling coatings [28], [29], has suitable properties for the “grafting to” method, in particular a low glass transition temperature (−10 °C) and a relatively low melting temperature (70 °C). Moreover, it can be prepared with high molecular uniformity and with a variety of terminal groups [30]. PEO brushes with high chain densities above 1 chain/nm2 (167 pmol/cm2) were prepared on a 2.5-nm-thick PGMA layer with PEO grafts of MW 5000 [31], [32]. Assuming that a great part of the terminal groups in this PEO layer can be modified with the cell–adhesion ligands, the resulting ligand surface density would well cover the range of 10−1–102 pmol/cm2, which has been reported to elicit cell adhesion and spreading on ultrathin hydrophilic polymer coatings [11], [15], [18], [33], [34], [35], [36]. The employment of PEO brushes prepared by the “grafting to” method for cell–ligand interaction studies therefore seems to be highly attractive, due to the substrate-independency and straightforward preparation based on ready-made polymers, simple casting and thermal procedures.

The goal of our work was to evaluate PEO brushes prepared by grafting PEO from melt to a PGMA anchoring layer as a platform for cell receptor–ligand engagement studies. The brushes were prepared from linear PEO carrying terminal alkynyl groups for subsequent modification with cell–adhesion ligands via azide–alkyne cycloaddition. We demonstrate that PEO/PGMA coating on silicon is sufficiently stable in an aqueous environment for several days, and is highly resistant to non-specific protein adsorption. Finally, coatings functionalized with RGD integrin ligands were shown to promote cell adhesion that is dependent on ligand surface density.

Section snippets

Polymers and reagents

Heterobifunctional PEOs α-(2-aminoethyl)-ω-methoxy-poly(ethylene oxide) (mPEO-NH2) and α-(2-aminoethyl)-ω-(2-(but-3-ynoylamino)ethoxy)-poly(ethylene oxide) (alkyne-PEO-NH2)(Rapp Polymere GmbH) were of Mn 5000 (Mp 4770, MALDI), and PDI of 1.1. Poly(glycidyl methacrylate) (PGMA), Mn 88,000, PDI 2.4 (PS calibration) was prepared by free radical polymerization. A mixture of 24 g of distilled glycidyl methacrylate, 36 ml of dry 1,4-dioxane and 240 mg of 2,2′-Azobis(2-methylpropionitrile) was purged

PGMA anchoring layer

To enhance covalent anchoring to the substrate, the PGMA layer was annealed in a vacuum at 110 °C [31]. A side effect of PGMA annealing is self-cross-linking, which is promoted by residual OH groups of hydrolyzed epoxides and which stabilizes the layer structure [31]. The extent of the covalent anchoring, self-cross-linking and the amount of epoxy groups entering the grafting process are parameters of high interest for explaining the properties of PGMA-based coatings. These properties have been

Conclusions

We have investigated the preparation and use of PEO/PGMA coatings as a non-fouling platform for specific ligand–cell receptor interaction studies. The kinetics of grafting amino-terminated PEO from melt to a PGMA anchoring layer revealed that the thickness of the grafted PEO layer depends not only on the grafting time but also largely on the thickness of the PGMA layer, due to the partial interpenetration of the two polymers. PEO/PGMA coatings with PEO layers thinner than 30 nm deposited on a

Acknowledgements

This work was supported by the Czech Science Foundation (Grant Nos. P207/10/P569 and P108/11/1857), and by BIOCEV – the Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University (CZ.1.05/1.1.00/02.0109), supported by the European Regional Development Fund. The authors would like to thank Mrs Z. Doubková and Mrs M. Brunclíková for technical assistance.

References (68)

  • M. Kothe et al.

    Examination of poly(butadiene epoxide)-coatings on inorganic surfaces

    Colloids Surf, A

    (1999)
  • J.H. Lee et al.

    Blood compatibility of polyethylene oxide surfaces

    Prog Polym Sci

    (1995)
  • U. Hersel et al.

    RGD modified polymers: biomaterials for stimulated cell adhesion and beyond

    Biomaterials

    (2003)
  • T.A. Petrie et al.

    The effect of integrin-specific bioactive coatings on tissue healing and implant osseointegration

    Biomaterials

    (2008)
  • I.M. Sapic et al.

    DFT study of molecular structure and vibrations of 3-glycidoxypropyltrimethoxysilane

    Spectrochim Acta Part A Mol Biomol Spectrosc

    (2009)
  • J. Búrdalo et al.

    A simple method to determine unperturbed dimensions of polymers using size exclusion chromatography and multiangle light scattering

    Polymer

    (2000)
  • L.T. Zhuravlev

    The surface chemistry of amorphous silica. Zhuravlev model

    Colloids Surf, A: Physicochem Eng Aspects

    (2000)
  • V.V. Krongauz et al.

    Imaging in evaluation of polymer coatings adhesion to glass at high humidity

    Prog Org Coat

    (1995)
  • J.C. Linehan et al.

    A simple determination of alkylsilane monolayer population density

    Inorg Chem Commun

    (2006)
  • B.T. Houseman et al.

    The microenvironment of immobilized Arg–Gly–Asp peptides is an important determinant of cell adhesion

    Biomaterials

    (2001)
  • M. Ventre et al.

    Determinants of cell-material crosstalk at the interface. Towards engineering of cell instructive materials

    J R Soc Interface

    (2012)
  • S.R. Meyers et al.

    Biocompatible and bioactive surface modifications for prolonged in vivo efficacy

    Chem Rev

    (2011)
  • G.A. Hudalla et al.

    Chemically well-defined self-assembled monolayers for cell culture: toward mimicking the natural ECM

    Soft Matter

    (2011)
  • G.A. Harbers et al.

    Development and characterization of a high-throughput system for assessing cell-surface receptor–ligand engagement

    Langmuir

    (2005)
  • G.M. Harbers et al.

    Functionalized poly(ethylene glycol)-based bioassay surface chemistry that facilitates bio-immobilization and inhibits nonspecific protein, bacterial, and mammalian cell adhesion

    Chem Mater

    (2007)
  • J. Groll et al.

    A novel star PEG-derived surface coating for specific cell adhesion

    J Biomed Mater Res, Part A

    (2005)
  • B.P. Harris et al.

    Photopatterned polymer brushes promoting cell adhesion gradients

    Langmuir

    (2006)
  • J.E. Raynor et al.

    Controlling cell adhesion to titanium: Functionalization of poly oligo(ethylene glycol)methacrylate brushes with cell-adhesive peptides

    Adv Mater

    (2007)
  • R. Barbey et al.

    Polymer brushes via surface-initiated controlled radical polymerization: synthesis, characterization, properties, and applications

    Chem Rev

    (2009)
  • B. Zdyrko et al.

    Polymer brushes by the “grafting to” method

    Macromol Rapid Commun

    (2011)
  • B. Zdyrko et al.

    Nano-patterning with polymer brushes via solvent-assisted polymer grafting

    Soft Matter

    (2008)
  • B.R. Coad et al.

    Substrate-independent method for growing and modulating the density of polymer brushes from surfaces by ATRP

    ACS Appl Mater Interfaces

    (2012)
  • O. Pop-Georgievski et al.

    Poly(ethylene oxide) layers grafted to dopamine–melanin anchoring layer: stability and resistance to protein adsorption

    Biomacromolecules

    (2011)
  • K.S. Iyer et al.

    Polystyrene layers grafted to macromolecular anchoring layer

    Macromolecules

    (2003)
  • Cited by (0)

    View full text