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