Elsevier

Biomaterials

Volume 26, Issue 15, May 2005, Pages 2333-2341
Biomaterials

Mediating specific cell adhesion to low-adhesive diblock copolymers by instant modification with cyclic RGD peptides

https://doi.org/10.1016/j.biomaterials.2004.07.010Get rights and content

Abstract

One promising strategy to control the interactions between biomaterial surfaces and attaching cells involves the covalent grafting of adhesion peptides to polymers on which protein adsorption, which mediates unspecific cell adhesion, is essentially suppressed. This study demonstrates a surface modification concept for the covalent anchoring of RGD peptides to reactive diblock copolymers based on monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) (H2N-PEG-PLA). Films of both the amine-reactive (ST-NH-PEG2PLA20) and the thiol-reactive derivative (MP-NH-PEG2PLA40) were modified with cyclic αvβ3/αvβ5 integrin subtype specific RGD peptides simply by incubation of the films with buffered solutions of the peptides. Human osteoblasts known to express these integrins were used to determine cell–polymer interactions. The adhesion experiments revealed significantly increased cell numbers and cell spreading on the RGD-modified surfaces mediated by RGD–integrin-interactions.

Introduction

The surface design of biomaterials applied in implant technology and tissue engineering is a key element in controlling the interaction with attaching cells and the surrounding tissue [1], [2], [3], [4]. Cell adhesion to a material is primarily mediated by integrins, with a plethora of integrin subtypes providing selective interactions with different proteins of the extracellular matrix [5], [6]. A number of integrin subtypes recognize the simple tripeptide sequence Arg-Gly-Asp (RGD) as their ligand, but show specific interactions depending on the amino acids flanking the RGD motif as well as on the conformation of the peptide [7], [8], [9]. The covalent linking of these adhesion peptides to biomaterials is a widely accepted approach to improve a material's biocompatibility, biological activity and its interactions with cells [9], [10], [11], [12], [13]. Biomaterials used in such attempts preferably suppress protein adsorption and the accompanying unspecific cell adhesion on their surfaces in order to provide undisturbed peptide dependent cell–biomaterial interactions [14]. Additionally, these materials need to provide a functional group to allow for the attachment of RGD peptides. Some hydrogels have been shown to be capable of fulfilling both specifications [12], [15], [16], [17], but lack the mechanical strength and macroporous structure necessary for many tissue engineering applications. Alternatively, non-swelling, lipophilic polymers have been investigated as materials for the fabrication of implants or scaffolds in the engineering of hard tissue. They provide higher mechanical strength and lower solubility in water, allowing for defined geometries, macroporosity and permeability [18], [19]. However, the covalent modification with RGD peptides is often a laborious procedure since most of these lipophilic polymers lack the required functional groups for surface modification [9]. To address the problem of unspecific protein adsorption and cell adhesion to lipophilic polymer surfaces, diblock copolymers, such as poly(D,L-lactic acid)-poly(ethylene glycol)-monomethyl ether (MeO-PEG-PLA), were developed. These polymers consist of both a hydrophilic and lipophilic chain and therefore combine mechanical stability and water insolubility with low adhesive properties. MeO-PEG-PLAs that contain high ratios of PEG, making them non-conductive for protein and peptide adsorption, have been particularly effective as ‘stealth’ biomaterials in numerous applications [20], [21]. We have recently shown that protein adsorption and cell adhesion can be controlled via the length and content of the MeO-PEG block in these diblock copolymers [21], [22]. To enable the convenient modification of these materials with adhesion peptides, mono amine derivatives (H2N-PEG-PLA) of the MeO-PEG-PLA diblock copolymers have been synthesized and subsequently functionalized by the covalent attachment of disuccinimidyl tartrate or N-succinimidyl-3-maleimido propionate, resulting in amine- (ST-NH-PEG-PLA) and thiol-reactive polymers (MP-NH-PEG-PLA), respectively [23], [24]. These reactive copolymers (Fig. 1) are designed to covalently bind peptides or proteins from aqueous solutions to preformed polymer surfaces during a simple incubation step. This concept would allow for an “of the shelf” scaffold or implant coating fabrication, which could be covalently modified with peptides in response to the individual needs by incubation with a sterile solution of the required peptide. The general feasibility of this concept has been shown in previous studies on the immobilization of fluorescent dyes or model proteins to preformed films and scaffolds [24], [25]. In the present study, we aimed at surface modification of reactive polymer films with αvβ5/αvβ3 integrin subtype specific cyclic RGD peptides via a free amine or thiol group (Fig. 2) [26], [27]. The success of the procedure was demonstrated in a cell adhesion study, since cell adhesion to and spreading on the modified low-adhesive surfaces requires a high density of integrin-binding sites [1], [28].

Two polymers were investigated in this study, ST-NH-PEG2PLA20, an amine-reactive copolymer composed of a 20 kDa PLA chain and a 2 kDa PEG chain, and MP-NH-PEG2PLA40, a thiol-reactive copolymer with a 40 kDa PLA chain. Polymer films were cast on glass object slides and subsequently modified with cyclic RGD peptides [27]. According to the polymer design concept, the RGD peptide was attached to the polymer surface by an instant procedure. This means that the prefabricated polymer films were simply incubated with a buffered solution of the peptide in a procedure adjusted to the chemistry of the polymers functional groups and to solid phase modification. The adhesion of human osteoblasts on the modified films was investigated, since this cell type is known to strongly express the corresponding αvβ5/αvβ3 integrin [5], [29]. This study demonstrates a surface modification concept based on low-adhesive, amine- and thiol-reactive copolymers, to which cell adhesion is mediated by covalently attached integrin subtype specific RGD peptides.

Section snippets

Polymer synthesis and characterization

The amine-reactive polymer ST-NH-PEG2PLA20 (Mw: 22 kDa) (α-Hydro-ω-[3-succinmidyl-oxycarbonyl-2,3-hydroxy-propyl-amido]-poly(oxy-1-oxopropane-2,1-diyl-block-oxyethylene)) (Fig. 1a) was synthesized from H2N-PEG2PLA20 and disuccinimidyl tartrate. Synthesis and analytical characterization were performed as described in the literature [23]. The synthesis and characterization of the thiol-reactive diblock copolymer MP-NH-PEG2PLA40 (Mw: 42 kDa)

Modification of the amine-reactive ST-NH-PEG2PLA20 films

Since we intended to covalently bind the RGD peptides from low-concentrated aqueous solutions, reaction conditions had to favor aminolysis of the polymer's N-hydroxysuccinimide ester rather than hydrolysis. Therefore, the peptide sequences were dissolved in a sodium bicarbonate buffer at pH 8 to ensure the presence of a neutral ε-amino-group on the lysine side chain for coupling with the N-hydroxysuccinimide ester, while the nucleophilicity of the arginine side chain in the cyclic RGD peptide

Conclusion

In conclusion, we showed the instant surface modification of preformed polymer films with high affinity adhesion peptides via two different linkers. The modification of low adhesive polymer films was performed by the simple incubation of amine- and thiol-reactive diblock copolymers, consisting of a reactive PEG and a PLA block. Although unspecific cell adhesion was shown to depend on the PEG content of the diblock copolymers, even MP-NH-PEG2PLA40 (5% PEG) displayed a sufficient concentration

Acknowledgements

The authors thank the Bayerische Forschungsstiftung for their financial support (ForTePro). Special thanks are due to Allison Dennis, Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA, for proof-reading.

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