Article pubs.acs.org/Biomac
Poly(2-oxazoline) Hydrogels for Controlled Fibroblast Attachment Brooke L. Farrugia,†,∥ Kristian Kempe,‡,▽ Ulrich S. Schubert,*,‡,§ Richard Hoogenboom,*,⊥ and Tim R. Dargaville*,† †
Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, 4059 Queensland, Australia ‡ Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstrasse 10, 07743 Jena, Germany § Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany ⊥ Supramolecular Chemistry Group, Department of Organic Chemistry, Ghent University, Krijgslaan 281 S4, B-9000 Ghent, Belgium S Supporting Information *
ABSTRACT: Currently there is a lack of choice when selecting synthetic materials with the cell-instructive properties demanded by modern biomaterials. The purpose of this study was to investigate the attachment of cells onto hydrogels prepared from poly(2-oxazoline)s selectively functionalized with cell adhesion motifs. A water-soluble macromer based on the microwave-assisted cationic ring-opening polymerization of 2-methyl-2-oxazoline and 2-(dec-9-enyl)-2-oxazoline was functionalized with the peptide CRGDSG or controls using thiol−ene photochemistry followed by facile cross-linking in the presence of a dithiol cross-linker. The growth of human fibroblasts on the hydrogel surfaces was dictated by the structure and amount of incorporated peptide. Controls without any peptide showed resistance to cellular attachment. The benignity of the cross-linking conditions was demonstrated by the incorporation of fibroblasts within the hydrogels to produce three-dimensional cell−polymer constructs.
1. INTRODUCTION The synthesis of cross-linked hydrophilic synthetic polymers using nontoxic conditions has inspired a large range of biomedical innovations combining polymers and proteins or cells in applications including drug delivery, tissue formation or regeneration, and substrates for studying cell-material interactions in three dimensions.1−3 Of particular interest are polymer matrices that can be used to support cell growth in a way that is controllable with respect to the physical and chemical cues provided to the cells within the microenvironment. A biomaterial approach that has proven to be popular in controlling the cell microenvironment is the use of a passive polymer serving as the cell support decorated with receptor ligands to provide the necessary regulating cues.4,5 In this context, the passivity is reflected by the ability to resist nonspecific protein adsorption, which can otherwise lead to presentation of the proteins in a way that is not observed naturally in vivo or may illicit an undesirable immune response.6 Poly(ethylene glycol) (PEG) is currently the “gold standard” in protein-resistant polymers and has attained universal acceptance. A significant drawback of PEG, however, is that it lacks chemical versatility and functionalization is typically limited to the end groups, meaning that the number of modifications possible per chain is small. Hence there is interest in polymers that have similar properties to PEG but can be synthesized © XXXX American Chemical Society
simply and contain a higher degree of functionality allowing for further modification. Poly(2-oxazoline)s (POxs) have recently been proposed as promising alternatives to PEG.7−10 This is partially based on the findings that surfaces coated with poly(2-methyl-2-oxazoline) (PMeOx) resist protein adsorption from human serum to an extent equal to or better than PEG.11 Similar resistance to bovine serum albumin was observed for thin films of poly(2ethyl-2-oxazoline) (PEtOx) on silicon and gold surfaces provided that the molar mass of the polymer was sufficiently high.12 PEtOx is a cheap commodity polymer, that is, Aquazol from Polymer Chemistry Innovations with broad molar mass distribution, synthesized via the cationic ring-opening polymerization (CROP) of 2-ethyl-2-oxazoline monomer. While PEtOx has many similarities to PEG, such as protein resistance and hydrophilicity, well-defined poly(2-oxazoline) (POx) has arguably greater versatility for structural modification through simple copolymerization or end group functionalization via judicious choice of initiator or terminating agent.13 This has led to many studies exploiting different POx architectures to Received: April 22, 2013 Revised: July 10, 2013
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dx.doi.org/10.1021/bm400518h | Biomacromolecules XXXX, XXX, XXX−XXX
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ether. 1H NMR and GPC data can be found in Figure S1 of the Supporting Information. 2.3. Hydrogel Preparation. Hydrogels were prepared from 10% w/v polymer solutions in doubly distilled water (ddH 2 O). Optimization of the UV irradiation time and photoinitiator concentration was carried out comparing initiator amounts ranging from 0% to 0.5% w/v and irradiation times up to 480 s. The light source was an Omnicure S1000 UV system with light guide, 365 nm filter, and OmniCure R2000 radiometer for calibration. The degree of peptide coupling to the POx copolymer was based on the equivalence of reacting alkene bonds. The monothiols, CRGDSG, CRDGSG, and 2-mercaptoethanol, were coupled to 0%, 5%, 10%, or 25% of the alkene groups present in the DecEnOx units leaving the remaining alkene bonds available to form cross-links with the dithiol, DTT. A typical hydrogel preparation protocol with 5% CRGDSG loading is as follows: to P(MeOx200-co-DecEnOx10) (14 mg in 70 μL of ddH2O, 0.73 μM polymer, 7.3 μM alkene bonds) were added 2.17 μL of a 10% w/v CRGDSG in ddH2O solution (equivalent to 0.22 mg, 0.37 μM), 1.4 μL of a 0.05% w/v solution of Irgacure 2959, and 61.07 μL of ddH2O. This mixture was irradiated for 240 s to conjugate the peptide. Subsequently, 5.36 μL of 10% w/v DTT solution (3.5 μM, 7.0 μM thiol groups) was added. Three aliquots of 20 μL each were pipetted onto a glass microscope coverslip pretreated with Sigmacote and sandwiched with a second coverslip using 0.97 mm spacers and irradiated for 480 s at RT. Following the curing step, hydrogels were immersed in ddH2O overnight at RT and then dried to constant weight at 60 °C. The xerogels were weighed (wdry) and then placed in phosphate-buffered saline (PBS) prewarmed to 37 °C. At predetermined time points, the swollen gels were removed from the PBS, blotted to remove any excess liquid, and weighed (wsw). The mass (Qm) and volumetric (Q) swelling ratios were determined using eqs 1 and 2: wsw − wdry Qm = wdry (1)
achieve self-assembling structures, whereas covalently crosslinked networks have received relatively little attention.14,15 Of the limited research examining cross-linked POx, the pioneering studies were reported by the group of Seagusa who cross-linked partially hydrolyzed PMeOx using a variety of reversible and nonreversible methods or cross-linked through the use of bis-2-oxazoline comonomers.16−21 Some of these studies were performed in aqueous conditions, but no biological applications were considered. More recently, crosslinking of PEtOx via γ-irradiation22 and microwave-assisted copolymerization with bis-2-oxazoline23 has been reported although neither of these approaches are suitable for encapsulation of cells or for use with fragile proteins. Other cross-linking approaches using POx as a copolymer or blend have been recently reviewed by Kelly and Wiesbrock.24 If POx is to be used as a support matrix for cells, it must be easily modified with groups to provide the necessary niche cues. The tripeptide arginine-glycine-aspartic acid (RGD) is a cell adhesion-mediating sequence found in fibronectin, laminin, and collagen and represents a popular choice for conjugation to polymers to improve the cell attachment. While there are many examples of POx−protein conjugates in the literature,25,26 the covalent attachment of RGD to POx has been reported just once before, by Luxenhofer et al., who used an alkyne group in the side chain of a PEtOx copolymer to react with azidefunctionalized cyclic RGD via a copper-catalyzed azide−alkyne cycloaddition but did not include any cell studies.27 Despite the variety of cross-linking methods reported for POx, the direct formation of hydrogels under aqueous conditions using mild chemicals remains largely unexplored, and to the best of our knowledge there are no studies using POx hydrogels in direct contact with cells. To extend the biomaterial applications of POx, we sought to use it as a platform to develop a fully synthetic, nontoxic, protein- and cell-compatible hydrogel system from readily available cheap materials with potential to be translated into cell culture laboratories. A recent review of the compatibility of POx with living systems has highlighted the suitability of PEtOx, PMeOx, and numerous copolymers as nontoxic biomaterials based on in vitro and animal studies.28 In the review, it is suggested that human trials are imminent and argued that once POx achieves regulatory approval and highquality commercial polymers become available, many more applications will follow. Novel POx networks cured under benign aqueous conditions are, therefore, an extremely attractive area to investigate for biomaterial applications.
Q=
Q m/ρsolvent + 1/ρpolymer 1/ρpolymer
(2)
where ρsolvent is the density of water (1.008 g/mL) and ρpolymer is the density of the polymer, estimated to be the same as that for PEtOx, namely, 1.14 g/mL. Dynamic mechanical analysis was conducted in frequency sweep mode using a DMA/SDTA 861e from Mettler-Toledo (Switzerland). A small clamp assembly containing the special shear clamp for low viscosity liquids was used to clamp a sample with a diameter of 5 mm. Distance screws made it possible to adjust the thickness of the sample to 0.325 mm enabling reproducible measurements. The force amplitude for the measurements was 1 N, and the displacement amplitude was 20 μm. Measurements were performed at 25 °C by applying a frequency sweep from 0.01 Hz up to 100 Hz with 20 steps per decade resulting in log G′ vs frequency plots (Figure S3 in the Supporting Information). At least three samples and four sweeps were measured per hydrogel composition. The average of these results is reported. 2.4. Fibroblast Attachment onto POx Hydrogels. Human dermal fibroblasts were isolated from consenting patients undergoing elective abdominal or breast reduction surgery. Ethics approval was obtained from the Queensland University of Technology’s Review Board (3673H) and associated hospitals, along with written informed consent from patients before undergoing surgery. In addition, the studies were conducted in strict accordance with the Declaration of Helsinki Principles. Dermal fibroblasts were isolated as described previously.30 POx hydrogels containing CRGDSG, CRDGSG, or 2mercaptoethanol were prepared as described above, then washed in ddH2O overnight, and sterilized by immersion in 70% EtOH for 3 h. Following sterilization, they were washed in PBS (3 × 1 h) and then placed in Dulbecco’s modified Eagle medium (DMEM, serum-free) overnight in 24 well plates. Isolated fibroblasts, 3 × 104 in 10 μL, were seeded onto the hydrogels and allowed to attach for 1 h, followed by
2. MATERIALS AND METHODS 2.1. Materials. The peptides CRGDSG, CRDGSG, and CRGDSCG were synthesized by Mimotopes Pty Ltd. (Melbourne, Australia) with free amine and acid end groups at purity >95% (determined by HPLC). Dithiothreitol (DTT, Roche), 2-mercaptoethanol (Sigma), and Irgacure 2959 (1-[4-(2-hydroxyethoxy)-phenyl]2-hydroxy-2-methyl-1-propane-1-one) (BASF) were used as-received. 2.2. Synthesis of P(MeOx-co-DecEnOx). Poly(2-methyl-2oxazoline-co-2-(dec-9-enyl)-2-oxazoline) (P(MeOx-co-DecEnOx)) was synthesized according to a previously reported method.29 Briefly, a solution of methyl tosylate (17 mg, 0.09 mmol), 2-methyl-2oxazoline (MeOx; 1.48 g, 17.4 mmol) and 2-(dec-9-enyl)-2-oxazoline (DecEnOx; 205 mg, 1 mmol) was prepared in acetonitrile (1.81 g, 44.1 mmol). The polymerization vial was heated to 140 °C in the microwave synthesizer for 20 min to reach near quantitative conversion. The resulting polymer was precipitated in ice-cold diethyl B
dx.doi.org/10.1021/bm400518h | Biomacromolecules XXXX, XXX, XXX−XXX
Biomacromolecules
Article
the addition of 1 mL of medium (10% FCS). Cells were cultured on the hydrogels for a period of 3 days prior to analyzing fibroblast attachment and morphology. Following cell culture, samples were fixed in 4% paraformaldehyde overnight at 4 °C then washed in PBS (3×). Fibroblasts were permeabilized in 0.2% Triton X for 5 min at RT, followed by blocking of nonspecific binding in 1% BSA for 1 h at 37 °C. Samples were incubated in 0.5 μg/mL 4′,6-diamidino-2phenylindole (DAPI, Invitrogen) for 5 min at RT and 200 U/mL Alexa Fluor 488 conjugated phalloidin (Invitrogen) for 20 min at RT. Following washing with PBS, the samples were imaged using a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) at λEx 495 nm and λEm 535 nm for visualization of f-actin and λEx 358 nm and λEm 641 nm for the nuclei. Cell morphology was quantified using Image J analysis of the actin orientation.31 2.5. Photoencapsulation of Fibroblasts. A series of P(MeOxco-DecEnOx) hydrogels was prepared incorporating fibroblasts within the matrix by cross-linking in the presence of cells. A combination of a bis-cysteine peptide (CRGDSCG) and DTT were used to cross-link the polymers, with a peptide to DTT ratio of 0.00:1.00, 0.05:0.95, 0.10:0.90, or 0.25:0.75. The 10% w/v P(MeOx-co-DecEnOx) polymer precursor solutions were prepared as described above but with ddH2O replaced with PBS. Confluent fibroblasts were trypsinized, centrifuged, and resuspended in the polymer/PBS solutions and then DTT, photoinitiator, and CRGDSCG were added followed by irradiation at 365 nm for 480 s in a laminar flow hood. The number of cells in each gel was 5 × 103 per gel. The resulting hydrogels were immediately placed into a 24 well plate containing DMEM (with 10% FCS) and cultured for either 1 or 8 days. Fibroblast viability was determined using fluorescein diacetate (FDA, Invitrogen) and propidium iodide (PI, Sigma Aldrich) stains by washing in PBS (containing Ca2+ and Mg2+), incubation in FDA (10 μg/mL) and PI (5 μg/mL) for 5 min at 37 °C, and finally washing in PBS. Samples were then imaged using confocal laser scanning microscopy.
Scheme 1. Reaction Showing the CROP of 2-Methyl-2oxazoline and 2-(Dec-9-enyl)-2-oxazoline Initiated with Methyl Tosylate and Terminated with Water
cationic ring-opening polymerization (CROP) to produce the polymer in under 20 min.29,36 To cross-link this polymer, we selected the photoinitiated thiol−ene reaction because it is known to tolerate the conditions used in biological systems including water and oxygen, and allows flexibility for inclusion of other molecules.37 Furthermore, the use of light to initiate the cross-linking is advantageous because it is highly accepted in medical and cell-culture laboratories. A water-soluble dithiol, namely, dithiothreitol (DTT) was chosen as the cross-linking agent, and Irgacure 2959 (I2959) was chosen as the photoinitiator. I2959 is sparingly soluble in water but with mild heating solutions of
