Thermoresponsive poly[tri(ethylene glycol) monoethyl ether methacrylate]-peptide surfaces obtained by radiation grafting-synthesis and characterisation
Graphical abstract
Introduction
The grafting of polymers to a solid surface provides a versatile tool for surface modification and functionalisation [1], [2], [3], [4]. Polymers can be grafted to the surface using two main approaches, grafting to or grafting from. Grafting to relies on the covalent attachment of polymer chains containing functional groups to the solid substrate. Grafting from involves polymerisation from the surface containing the appropriate initiating groups. To obtain polymer layers by the grafting from method, many polymerisation techniques, such as living ionic polymerisation [5], reversible addition−fragmentation chain transfer polymerisation [6], atom transfer radical polymerisation (ATRP) [7], nitroxide-mediated radical polymerisation [8] and radiation grafting [9], [10], [11], [12], [13], [14], [15] have been used. The latter method has been extensively tested in manufacturing materials for applications, such as separators in batteries and fuel cells, adsorbents for the removal of pollutants or rare metal ions, as well as hydrophilic and/or stimuli-sensitive surfaces for biomaterials [16], [17]. Some of these materials are now commercially available [18].
Radiation grafting can be performed either in a direct (simultaneous) way, where the substrate and monomer are irradiated together, or as an indirect (post-irradiation) process, where only the substrate is irradiated to generate reactive species, and it is later contacted with the monomer in the absence of radiation. In the latter case, if substrate irradiation is performed in the presence of oxygen, the reactive species formed during irradiation are mainly peroxyl and hydroperoxyl groups. When the substrate containing these reactive entities is contacted, preferably in the absence of oxygen, with a monomer or monomer solution at elevated temperatures (typically 60–80 °C), peroxides and hydroperoxides are thermally decomposed forming oxygen-centred radicals, mostly of the oxyl type, which initiate graft polymerisation. This technique has been applied to obtain e.g. thermoresponsive surfaces of polyvinylidene fluoride-graft-poly(N-isopropylacrylamide) [19], polypropylene-graft-poly(triethyleneglycol ethyl ether methacrylate) [20], poly(di(ethyleneglycol) methyl ether methacrylate) and poly(oligo(ethylene glycol) methyl ether methacrylate)s (Mn values of oligo(ethylene glycol) methyl ether methacrylate monomers were 300 and 475, respectively) with different monomer concentrations and molar ratios [21]. However, much less is known about the possibility of incorporating biologically active compounds into the grafted thermoresponsive layer during the radiation grafting process, in such a way that these compounds become the covalently bound element of the graft.
The use of thermoresponsive polymeric surfaces as scaffolds for harvesting cell sheets and detachment is highly desirable in cell sheet engineering [22]. Such surfaces in aqueous environments change their properties in response to temperature. Below a certain temperature (the cloud point temperature, TCP), polymer chains are hydrated by water molecules and become swollen. When heated above TCP, polymer chains undergo a phase transition which leads to their dehydration and shrinkage [23]. This behaviour results in a fast switch in surface philicity from hydrophobic (cell attractive) to hydrophilic (cell repellent) in a manner that is completely reversible.
The most studied thermoresponsive polymeric surface for cell sheet engineering applications is poly(N-isopropylacrylamide) (PNIPAM) and its copolymers. PNIPAM is a well characterised thermoresponsive polymer that undergoes a sharp phase transition in aqueous solution at a temperature around 32 °C [24].
Promising alternatives for PNIPAM are poly(oligo(ethylene glycol) methacrylates) (POEGMAs). The heating and cooling behaviours of POEGMAs are similar, whereas PNIPAM solutions exhibit hysteresis [25]. Additionally, the TCP of POEGMAs in aqueous solutions can be tuned over a wide range of temperatures (20–90 °C) by simply changing the length of the oligo(ethylene glycol) side chains [25] or by the copolymerisation of different oligoethylene glycols [26], [27]. Moreover, POEGMAs have been shown to exhibit increased biocompatibility compared to PNIPAM [28].
It was shown that layers of poly(triethyleneglycol ethyl ether methacrylate) (PTEGMA) grafted from the glass surface by ATRP were successfully used to obtain confluent cell sheets of human fibroblast that can be easily and quickly separated as monolayers by simple decreasing the temperature below the TCP of the polymer [7].
Peptide sequences, such as RGDS (arginine-glycine-aspartic acid-serine), laminin-derived YIGSR (tyrosine-isoleucine-glycine-serine-arginine) and the fibrinogen derived IKVAV (isoleucine-lysine-valine-alanine-valine), have been used for the modification of many natural and synthetic materials to induce specific cell behaviours [29], [30], [31], [32], [33], [34]. These peptides are particularly important for functionalisation of scaffolds for cell culturing and harvesting.
The introduction of cell-adhesive peptides to thermoresponsive scaffolds was investigated by Okano [35], [36]. RGDS peptide was covalently attached to poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide)-grafted surfaces by a condensation reaction using a carbodiimide coupling reagent. Immobilisation of the RGDS peptide to temperature-responsive surfaces facilitates both serum-free cell adhesion and non-invasive cell harvesting [36].
In this work, we demonstrate the feasibility of indirect post-irradiation grafting using an electron beam to prepare thermoresponsive polymer layers of PTEGMA functionalised with short cell promoting CF-IKVAVK (CF-carboxyfluorescein, I-isoleucine, K-lysine, V-valine, A-alanine, K-lysine) peptide ligands on a polypropylene (PP) support. CF-IKVAVK was synthesised according to the Fmoc strategy of solid phase peptide synthesis. The peptide was modified with a methacrylamide group which is active in the polymerisation process and enabled its attachment to the polymer layer during the grafting process. The amount of CF-IKVAVK on the surface was easily controlled by changing its concentration in the reaction mixture. Several surface characterisation techniques, such as atomic force microscopy (AFM), contact angle, gravimetric analysis, secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS), were used to monitor the modification steps and to analyse the formed polymer layer. The presented synthetic strategy is versatile and may be adopted to obtain scaffolds for cell culturing and harvesting.
Section snippets
Materials
PP Petri-like dishes (Atix, Poland) of 35 mm diameter, 11 mm height and 0.8 mm thickness were soaked in ethanol for 15 min and dried at room temperature overnight. Tri(ethylene glycol) monoethyl ether methacrylate (TEGMA, M = 246 g/mol) was purchased from ECEM European Chemical Marketing and was used as received. Isopropanol for the polymerisation supplied by Eurochem was also used as received. Water was purified using a MicroPure setup (TKA, Germany, specific resistivity of 18.2 MΩ cm). The argon (Ar
Methods
Electrospray Ionisation-Mass Spectrometry was applied to analyse the peptide structure. The ESI–MS spectra of peptides were collected on an AmaZon ETD (Bruker Daltonics, Bremen, Germany) mass spectrometer. The mass spectrometer settings were as follows, mass spectra were registered in positive ion mode, a capillary voltage of −4500 V was used and the temperature of the heated capillary was 280 °C. The flow rate of the injected samples was 3 μL/min. The solvent used for the experiments consisted of
Conclusions
This work showed that radiation grafting can be used to obtain thermoresponsive PTEGMA surfaces containing the cell adhesion peptide, CF-IKVAVK. Radiation grafting is relatively simple and a versatile two-step procedure. Surface analysis using different techniques (ToF-SIMS, FTIR, XPS and AFM) revealed that this method leads to homogeneously spread polymer layers on the PP support. The amount of peptide attached to the surface can be easily controlled by changing its concentration in the
Acknowledgements
The authors thank Dr. Alicja K. Olejnik and Dr. Marian Wolszczak (IARC) for spectrofluorimetric analyses and the staff of the linear electron accelerator lab at IARC for their skilful technical assistance.
This work was financed by the National Centre for Research and Development, project POLYCELL PBS1/B9/10/2012. Roza Szweda was a scholarship holder within the DoktoRIS project-scholarship program for the innovation of the Silesia region supported by the European Community from the European
References (47)
- et al.
Polymer brushes––surface immobilized polymers
Surf. Sci.
(2004) - et al.
Radiation modification of silicone rubber with glycidylmethacrylate
Radiat. Phys. Chem.
(2013) - et al.
Functionalization of polymer surfaces by radiation-induced grafting for separation of heavy metal ions
Radiat. Phys. Chem.
(2014) - et al.
Mucoadhesive thermo-responsive chitosan-g-poly(N-isopropylacrylamide) polymeric micelles via a one-pot gamma-radiation-assisted pathway
Colloids Surf. B
(2015) - et al.
Synthesis of poly(sulfobetaine methacrylate)-grafted chitosan under-ray irradiation for alamethicin assembly
Colloids Surf. B
(2015) - et al.
Stimuli-reponsive polymers and their bioconjugates
Prog. Polym. Sci.
(2004) - et al.
Temperature-sensitive porous membrane production through radiation co-grafting of NIPAAm on/in PVDF porous membrane
Radiat. Phys. Chem.
(2007) - et al.
Radiation grafting of oligo(ethylene glycol) ethyl ether methacrylate on polypropylene
Radiat. Phys. Chem.
(2014) - et al.
Temperature-responsiveness and biocompatibility of DEGMA/OEGMA radiation-grafted onto PP and LDPE films
Radiat. Phys. Chem.
(2014) - et al.
Smart polymers: physical forms and bioengineering applications
Prog. Polym. Sci.
(2007)