Biomacromolecules 2008, 9, 2265–2275
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Biocompatible Wound Dressings Based on Chemically Degradable Triblock Copolymer Hydrogels Jeppe Madsen,† Steven P. Armes,*,† Karima Bertal,‡ Hannah Lomas,‡ Sheila MacNeil,‡ and Andrew L. Lewis§ Dainton Building, Department of Chemistry, The University of Sheffield, Brook Hill, Sheffield, S3 7HF, United Kingdom, Department of Engineering Materials, The University of Sheffield, The Kroto Research Institute, Broad Lane, Sheffield, S3 7HQ, United Kingdom, and Biocompatibles UK Ltd., Chapman House, Farnham Business Park, Weydon Lane, Farnham, Surrey, GU9 8QL, United Kingdom Received May 6, 2008
The synthesis of a series of thermo-responsive ABA triblock copolymers in which the outer A blocks comprise poly(2-hydroxypropyl methacrylate) and the central B block is poly(2-(methacryloyloxy)ethyl phosphorylcholine) is achieved using atom transfer radical polymerization. These novel triblock copolymers form thermo-reversible physical gels with critical gelation temperatures and mechanical properties that are highly dependent on the copolymer composition and concentration. TEM studies on dried dilute copolymer solutions indicate the presence of colloidal aggregates, which is consistent with micellar gel structures. This hypothesis is consistent with the observation that incorporating a central disulfide bond within the B block leads to thermo-responsive gels that can be efficiently degraded using mild reductants such as dithiothreitol (DTT) over time scales of minutes at 37 °C. Moreover, the rate of gel dissolution increases at higher DTT/disulfide molar ratios. Finally, these copolymer gels are shown to be highly biocompatible. Only a modest reduction in proliferation was observed for monolayers of primary human dermal fibroblasts, with no evidence for cytotoxicity. Moreover, when placed directly on 3D tissue-engineered skin, these gels had no significant effect on cell viability. Thus, we suggest that these thermoresponsive biodegradable copolymer gels may have potential applications as wound dressings.
Introduction It is well-known that ABA triblock copolymers with watersoluble central B blocks and water-insoluble outer A blocks can form either flower micelles or free-standing gels in aqueous solution, depending on the copolymer concentration and the copolymer composition.1–8 The gels are of particular interest and comprise a three-dimensional network of interconnected micelles, with the water-soluble B blocks acting as bridges between adjacent micelles. Computer simulations indicate that critical copolymer volume fractions of 0.05-0.10 are required for gelation, depending on the overall molecular mass.3 This work and other theoretical studies1,2 also predict that network formation depends mainly on the molecular weights of both blocks, as well as the hydrophobic character of the outer A block. These findings have been confirmed by a number of experimental studies.1,6 We recently reported an example of a pH-responsive triblock gelator.9 Here the A blocks were based on poly(2-(diisopropylamino)ethyl methacrylate) [PDPA] and the central B block comprised poly(2-(methacryloyloxy)ethyl phosphorylcholine) [PMPC], a highly hydrophilic polymer with excellent biocompatibility.10,11 The PDPA chains become protonated below their pKa of around 6.3, which allows molecular dissolution of the triblock copolymer chains in acidic solution. Neutralization of this solution leads to free-standing gels. However, precise control over the in situ pH adjustment is somewhat problematic and biological actives such as cells, DNA, or proteins may not survive the initial acidic conditions. * To whom correspondence should be addressed. E-mail: armes@ sheffield.ac.uk. † Department of Chemistry, The University of Sheffield. ‡ Department of Engineering Materials, The University of Sheffield. § Biocompatibles UK Ltd.
Given these disadvantages, we designed a second-generation thermo-responsive triblock gelator12 in which the PDPA blocks were replaced with poly(N-isopropylacrylamide) [PNIPAM], a water-soluble polymer that is well-known for its inverse temperature solubility behavior.13,14 This modification allowed the formation of transparent free-standing gels at 37 °C, which were sufficiently biocompatible to allow in situ cell proliferation. Moreover, using a disulfide-based initiator allowed the synthesis of a third-generation PNIPAM-PMPC-PNIPAM triblock containing a single S-S bond within the backbone of the central PMPC block. Selective cleavage of this S-S bond using a naturally occurring tripeptide such as glutathione converted the triblock copolymer into a diblock copolymer of approximately half the original triblock molecular weight. Because the resulting PMPC-PNIPAM diblock copolymer cannot form free-standing gels, this led to the concept of a biochemically responsive gel based on a disulfide “keystone”. Unfortunately, NIPAM is not an ideal building block for the design of thermo-responsive copolymers for biomedical applications. This monomer is relatively expensive, requires purification prior to use, and is a potent neurotoxin.15 It is also not trivial to copolymerize NIPAM with methacrylic monomers such as MPC using atom transfer radical polymerization (ATRP),12,16 which is our preferred synthetic methodology. In view of this, we recently explored using several hydrophilic methacrylic monomers in place of NIPAM. We had already shown that 2-hydroxyethyl methacrylate [HEMA] exhibited thermo-responsive behavior in aqueous solution,17 but unfortunately, PHEMA-PMPC-PHEMA triblocks proved insufficiently hydrophobic to form free-standing gels. On the other hand, an analogous triblock copolymer based on 2-hydroxypropyl methacrylate [HPMA] instead of HEMA gave very encouraging preliminary results.18 Thus, a PHPMA50-
10.1021/bm8005006 CCC: $40.75 2008 American Chemical Society Published on Web 07/04/2008
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PMPC250-PHPMA50 triblock copolymer could be dissolved in cold water but formed free-standing gels at higher temperatures. Moreover, the critical gelation temperature was relatively sensitive to the copolymer concentration, which is not the case for PNIPAM-based copolymers. In the present work, we report our studies of a series of PHPMA-PMPC-PHPMA triblock copolymers of varying block composition and molecular weight. Their aqueous gelation behavior is examined using gel rheology, light scattering, variable temperature 1H NMR spectroscopy, and transmission electron microscopy, and the biocompatibility of selected gels was assessed for potential wound dressing applications.
Experimental Section Materials. 2-(Methacryloyloxy)ethyl phosphorylcholine monomer (MPC, 99.9% purity) was kindly donated by Biocompatibles U.K. Ltd. 2-Hydroxypropyl methacrylate (HPMA) was kindly donated by Cognis Performance Chemicals (Hythe, U.K.). Bis(2-hydroxyethyl)disulfide (98%), 2-bromoisobutyryl bromide (98%), basic alumina (Brockmann I, standard grade, ∼150 mesh, 58 Å), dithiothreitol (DTT, 99%), glutathione (99%), DL-cysteine (g95%), anhydrous methanol (MeOH 99.8%), copper(I) bromide [Cu(I)Br, 99.999%], 2,2′-bipyridine (bpy, 99%), and diethyl meso-2,5-dibromoadipate (DEDBA, 98%) were purchased from Sigma Aldrich UK and were used as received. The silica gel 60 (0.063-0.200 µm) used to remove the spent ATRP catalyst was purchased from E. Merck (Darmstadt, Germany) and was also used as received. Dubelco’s modified medium (DMEM), MTT, cholera toxin, epidermal growth factor, adenine, insulin, sodium chloride, triiodothyronine, and EDTA were all purchased from Sigma-Aldrich (Poole, Dorset, U.K.). Foetal calf serum (FCS) was purchased from Labtech (East Sussex, U.K.). Ham’s F12 medium, glutamine, amphotericin B, penicillin, and streptomycin were purchased from Gibco (Paisley, U.K.). Hydrocortisone was purchased from Novabiochem (Nottingham, U.K.). Trypsin was purchased from Difco Laboratories (Detroit, MI). Collagenase A was purchased from Boehringer-Mannheim (East Sussex, U.K.). ThinCerts were purchased from Greiner Bio-one (Stonehouse, U.K.). Stainless steel rings and grids were supplied by the Royal Hallamshire Hospital, (Sheffield, U.K.). Synthesis of the Disulfide-Based Bifunctional ATRP Initiator, Bis[2-(2-bromoisobutyryloxy)ethyl] Disulfide (BiBOE)2S2. The disulfide-based bifunctional ATRP initiator (BiBOE)2S2 was synthesized according to a literature protocol.19,20 Bis(2-hydroxyethyl) disulfide (15.4 g, 0.1 mol) was dissolved in 200 mL of dry THF, excess triethylamine (42.0 mL, 0.30 mol) was added under a nitrogen atmosphere, and this solution was cooled in an ice bath. 2-Bromoisobutyryl bromide (59.8 g, 0.26 mol) was added dropwise from a dropping funnel over a 1 h period so as to minimize the reaction exotherm, and the reaction solution slowly turned reddish brown. The solution was allowed to warm up to ambient temperature and stir for 24 h. The insoluble triethylammonium bromide salt was removed by filtration and the resulting colorless solution was concentrated under vacuum. The concentrated solution was stirred with 0.10 M aqueous Na2CO3 to hydrolyze any residual 2-bromoisobutyryl bromide. The crude product was then extracted three times with dichloromethane using a separating funnel. The combined dichloromethane extracts were first dried with anhydrous magnesium sulfate and then concentrated to afford a reddish brown oil (31.2 g; yield ) 69%), which was stored in a refrigerator prior to use. The crude product was purified by dissolution in dichloromethane, followed by passage through a basic alumina column to yield a pale yellow liquid that crystallized in the freezer (-25 °C). This (BiBOE)2S2 initiator was analyzed by 1H NMR spectroscopy in CDCl3 (δ, ppm): 4.45 (triplet, 2H, -CH2OOC-), 2.96 (triplet, 2H, -CH2S-), and 1.95 [singlet, 6H, (CH3)2C-]. Elemental microanalyses gave Br ) 35.33% (theory 35.4%) and S ) 13.96% (theory 14.2%), suggesting that the initiator purity exceeded 98%.
Madsen et al. Copolymer Synthesis and Purification. One-pot copolymer syntheses were conducted using sequential monomer addition without purification of the intermediate PMPC macro-initiator. A typical synthesis was carried out as follows: MPC (10.002 g, 33.9 mmol, 250 equiv) was placed under nitrogen. (BiBOE)2S2 (61.2 mg, 0.135 mmol, 1 equiv) was dissolved in 12 mL of anhydrous methanol and added to the MPC through a cannula. The reaction mixture was purged with nitrogen for 30 min. 2,2′-Bipyridine (83.8 mg, 0.537 mmol, 4.0 equiv) and Cu(I)Br (38.6 mg, 0.269 mmol, 2.0 equiv) were added to commence the first-stage polymerization. After 6 h, HPMA (1.9533 g, 13.5 mmol, 100 equiv) was added to the dark brown viscous solution by cannula and the reaction mixture was stirred for an additional 70-100 h until no vinyl signals were observed in the 1H NMR spectrum. After this time period, the reaction mixture was diluted with methanol and passed through a silica column to remove the spent catalyst. The solution was partly evaporated and precipitated into excess THF (500 mL) to remove residual monomer and traces of 2,2′-bipyridine. After filtration, residual THF was removed by coevaporation with three 50 mL portions of methanol. To the solid residue was added 200 mL of water and this was stirred until a uniform mixture was obtained. The water was evaporated at 50-60 °C under reduced pressure to obtain a solution volume of approximately 50 mL prior to addition of 150 mL of water. Approximately 150 mL of water was again removed under vacuum and the resulting solution was freeze-dried overnight. Finally, the copolymer was dried at 80 °C at high vacuum for 48 h, then for 5-6 h at 90 °C. These additional coevaporation steps were essential for the cell studies, because it was found that traces of cytotoxic methanol were very difficult to remove by simply drying the copolymer in a vacuum oven. In contrast, repeated coevaporation of residual methanol with water under reduced pressure proved to be a reliable means of ensuring sufficient purification to achieve biocompatibility. This protocol produced 9-10 g of purified triblock copolymer (75-83% yield). 1 H NMR Spectroscopy. 1H NMR spectra were recorded in either D2O or CD3OD using either a 400 MHz Bruker AV1-400 or a 500 MHz Bruker DRX-500 spectrometer. For the variable temperature studies, the integrated peak intensity due to the pendent methyl groups in the PHPMA chains at 1.3 ppm was compared to that due to the pendent methylene groups of the PMPC chains at 3.7 ppm. This numerical value was normalized with respect to the actual block composition of the copolymer, as determined by 1H NMR in CD3OD, which is a good solvent for both PHPMA and PMPC. Thus, the apparent block composition could be estimated at any given temperature. Molecular Weight Determination. Chromatograms were assessed using a Hewlett-Packard HP1090 Liquid Chromatograph as the pumping unit and two Polymer Laboratories PL Gel 5 µm Mixed-C (7.5 × 300 mm) columns in series with a guard column at 40 °C connected to a Gilson Model 131 refractive index detector. The eluent was a 3:1 v/v % chloroform/methanol mixture containing 2 mM LiBr at a flow rate of 1.0 mL min-1. A series of near-monodisperse poly(methyl methacrylate) [PMMA] samples were used as calibration standards. Toluene (2 µL) was added to all samples as a flow rate marker. Data analyses were conducted using CirrusTM GPC Software supplied by Polymer Laboratories. Dynamic Light Scattering. Copolymer solutions for light scattering studies were prepared as 1.00 wt % aqueous solutions in PBS. These stock solutions were diluted to the desired concentration and filtered through a 0.43 µm nylon filter prior to use. Dynamic light scattering experiments were performed with a Zetasizer Nano-ZS (Malvern Instruments, U.K.) at a scattering angle of 173 °. Dispersion Technology Software version 4.20, supplied by the manufacturer, was used for cumulants analysis according to ISO 13321:1996. Transmission Electron Microscopy. Samples were mounted on precoated carbon-coated copper grids. These grids were submerged for 1 min into a 0.40% aqueous copolymer solutions at 25 °C and then in an aqueous uranyl acetate solution (1% w/w) for 20 s. Imaging was
Biocompatible Wound Dressings performed on a FEI Tecnai Spirit TEM operating at 120 kV equipped with a Gatan 1K MS600CW CCD camera. Gel Rheology Studies. Copolymer (30.0-300.0 mg) was dissolved in aqueous PBS solution (1.00 mL) for rheology studies. These solutions were left to stand in a refrigerator at 4 °C overnight. For more concentrated copolymer solutions (10-30%, depending on the copolymer composition and its molecular weight), the solutions were subjected to several freeze-thaw cycles to remove trapped air. A Rheometric Scientific SR-5000 rheometer equipped with cone-plate geometry (40.0 mm, 0.05 radians) was used for the oscillatory temperature sweeps, employing a frequency of 1 rad/s, a stress of 0.5 Pa, and a heating rate of 3 °C/min. This instrument was fitted with a Peltier element for temperature control and a thermostatted water-bath was used as a heat sink. Gel Cleavage Experiments. Aqueous triblock copolymer solutions were prepared in PBS buffer that had been purged with nitrogen for several hours prior to use to exclude oxygen. Sample preparation was otherwise identical to that used for the temperature-sweep experiments. DTT concentrations were calculated assuming that the Mn of the PHPMA88-PMPC200-S-S-PMPC200-PHPMA88 was 150,000. Addition of the DTT reductant was achieved by placing 1.0 mL of a 11.0% copolymer solution and 0.10 mL of the reductant solution in two separate syringes. These syringes were connected by a three-way valve and thermostatted in a water bath at the desired temperature for 1.5 min. These solutions were then mixed for 10 s by pushing the plungers forward and back. The valve was opened and the resulting aqueous mixture of copolymer and reductant was placed in the thermostatted rheometer. Measurements commenced approximately 35-40 s after mixing using the cone-and-plate geometry. The applied stress was 0.06 Pa and the frequency was 1 rad per second. Cell Culture Studies. Skin was obtained from patients undergoing breast reductions and abdominoplasty elective surgical procedures. Patients gave informed consent for skin not required for their treatment to be used for experimental purposes under a protocol approved by the Ethical Committee of the Northern General Hospital Trust (NHS), Sheffield, U.K. Fibroblasts and keratinocytes were isolated from skin according to the methods described by Ghosh et al.21 Fibroblasts were isolated and cultured in DMEM, supplemented with 10% v/v FCS, 2 × 10-3 mol dm-3 glutamine, 0.625 µg/mL amphotericin B, 100 IU/mL penicillin, and 100 µg/mL streptomycin. For each experiment, passage 4-8 fibroblasts were used. Keratinocytes were isolated and cultured in Green’s medium containing: 108 mL of Ham’s F12 nutrient mixture per 500 mL, 330 mL DMEM per 500 mL, 10% v/v FCS, 2 × 10-3 mol dm-3 glutamine, 0.625 µg/mL amphotericin B, 100 IU/mL penicillin, 100 µg/mL streptomycin, 20 µg/mL adenine, 5 µg/mL insulin, 2 × 10-7 M triiodothyronine, 5 µg/mL transferrin, 0.4 µg/ mL hydrocortisone, 10 ng/mL epidermal growth factor (EGF), and 8.5 mg/mL cholera toxin. Freshly isolated keratinocytes were used in all experiments. Preparation of 3D Tissue-Engineered Skin. Tissue-engineered skin (TE skin) was prepared as detailed by Ghosh et al.,21 except that the dermis was not sterilized for the current experiments. Briefly, deepidermized dermis (DED) was prepared by incubating the skin in 1.0 M NaCl for 24 h at 37 °C to remove the epidermis. DED was then cut into 2 × 2 cm squares and placed in 6-well plates with the papillary surface oriented upward. A stainless steel ring was then placed on the top of the dermis to create a seal. A cell suspension containing 3 × 105 keratinocytes and 1 × 105 fibroblasts was then seeded onto the papillary surface of DED within the ring. Additional media was then added outside the ring to keep the DED moist. The ring was left in place for 48 h to allow cell attachment. The ring was then removed and the DED was raised to the air-liquid interface using stainless steel grids. TE constructs were cultured for 8 days at the air-liquid interface before exposing them to the gels. The medium was changed every 3-4 days.
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Effect of Copolymers on Cell Monolayers. Fibroblasts were cultured for 24 h in a humidified atmosphere of 5% CO2 at 37 °C at a density of 40,000 cells per well in 24-well plates. The 10% w/v and 20% w/v gels were prepared by dissolving PHPMA-PMPC-S-S-PMPCPHPMA copolymers into media, followed by incubation at 4 °C for 24 to 48 h until full dissolution of the copolymer was observed. The gels were then transferred into 24-well plate ThinCerts containing a welded poly(ethylene terephthalate) capillary pore membrane with a 0.8 µm pore diameter. These gel-loaded “baskets” were then suspended over the cells for 24-72 h (see Supporting Information Figure S4). An MTT assay was undertaken after 24, 48, or 72 h exposure to the copolymer gel to assess cell viability. Effect of Copolymers on 3D Tissue-Engineered Skin. The TE skin models were cultured for eight days at an air-liquid interface before being exposed to the copolymer gels. Copolymer (20% w/v) was dissolved in media and incubated for 48 h at 4 °C to ensure complete dissolution. Stainless steel rings (as shown in Figure S5, Supporting Information) were placed on the TE skin models to which the gel was added. These models were exposed to the gel for four days, after which an assessment of cell viability and structure was achieved using MTT assay and histology studies, respectively. Histology. Composites were transferred to 10% phosphate-buffered formaldehyde at room temperature. They were embedded in paraffin wax, and 5 µm sections were cut, mounted, and stained using hematoxylin and eosin. Assessment of Cell Viability Using the MTT-Eluted Stain Assay. Viable cell density on 2D monolayer/3D TE models was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The MTT salt is reduced by metabolically active (i.e., viable) cells into formazan via cellular dehydrogenase enzymes. The formazan is then solubilized and elutes the stain, whose density can be determined using a spectrophotometer operating at 540-630 nm. The optical density is proportional to the metabolic activity of the cells, reflecting thereby cell viability. Briefly, the cultured cells or tissues were washed thoroughly with PBS and then incubated in MTT solution (0.5 mg/mL MTT in PBS) for either 1 h (2D cell monolayer) or 2 h (3D TE skin) at 37 °C in 95% air/5% CO2. Subsequently, the solution was aspirated and the insoluble formazan product was solubilized with either acidified iso-propanol (2D cell monolayer) or 2-ethoxyethanol “cellusolve” (3D TE skin). The optical density was then determined at 540 nm using a plate reading spectrophotometer (MRX microplate reader). Statistical Analysis. Statistical analysis was performed using the paired Student t-test, in which a p-value of
