Mediating specific cell adhesion to low-adhesive diblock copolymers by instant modification with cyclic RGD peptides
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.
References (34)
- et al.
Arg-Gly-Asp constrained within cyclic pentapeptides. Strong and selective inhibitors of cell adhesion to vitronectin and laminin fragment P1
FEBS Lett
(1991) - et al.
RGD modified polymersbiomaterials for stimulated cell adhesion and beyond
Biomaterials
(2003) Osteoblast adhesion on biomaterials
Biomaterials
(2000)- et al.
Alginate hydrogels as synthetic extracellular matrix materials
Biomaterials
(1999) - et al.
Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering
Biomaterials
(2002) - et al.
In vitro generation of osteochondral composites
Biomaterials
(2000) - et al.
‘Stealth’ corona-core nanoparticles surface modified by polyethylene glycol (PEG)influences of the corona (PEG chain length and surface density) and of the core composition on phagocytic uptake and plasma protein adsorption
Colloids Surf B
(2000) - et al.
Biodegradable poly(lactic acid)-poly(ethylene glycol)-monomethyl ether diblock copolymersstructures and surface properties relevant to their use as biomaterials
Biomaterials
(2000) - et al.
The use of poly(ethylene glycol)-block-poly(lactic acid) derived copolymers for the rapid creation of biomimetic surfaces
Biomaterials
(2003) - et al.
Towards biomimetic scaffoldsAnhydrous scaffold fabrication from biodegradable amine-reactive diblock copolymers
Biomaterials
(2003)
Extracellular matrix cell adhesion peptidesfunctional applications in orthopedic materials
Tissue Eng
Creating biomimetic micro-environments with synthetic polymer-peptide hybrid molecules
J Biomater Sci Polym Ed
Porous polymer scaffolds surface-modified with arginine-glycine-aspartic acid-cysteine enhance bone cell attachment and differentiation in vitro
J Biomed Mater Res
Induced tissue integration of bone implants by coating with bone selective RGD-peptides in vitro and in vivo studies
J Mater Sci Mater Med
Integrin expression and function on human osteoblast-like cells
J Bone Miner Res
RGD and other recognition sequences for integrins
Annu Rev Cell Dev Biol
Structure and function of RGD peptides involved in bone biology
Cell Mol Life Sci
Cited by (61)
New perspectives: In-situ tissue engineering for bone repair scaffold
2020, Composites Part B: EngineeringThe Extracellular Matrix and Cell–Biomaterial Interactions
2020, Biomaterials Science: An Introduction to Materials in MedicineSurface modification of copolymerized films from three-armed biodegradable macromers – An analytical platform for modified tissue engineering scaffolds
2017, Acta BiomaterialiaCitation Excerpt :While the latter opens a platform for immobilization of other biotinylated effector molecules to the surfaces, a comparison of both methods was done to determine changes in activity of ALP by direct covalent immobilization. RGD peptides are a frequently used means to improve cell adhesion to non-adherent surfaces [27]. Knowledge about design parameters for such peptides [28] and conformation-related specificity is available [29].
CRGD-functionalized polymeric magnetic nanoparticles as a dual-drug delivery system for safe targeted cancer therapy
2013, Pharmacological Research
- 1
These authors contributed equally to the paper.