Synthesis and Characterization of Biocompatible, Thermoresponsive

Bowl- and porous sphere-shaped supramolecular assemblies and their application as templates for confined assembly of gold nanoparticles. Fang-Ming Xu ...
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Langmuir 2005, 21, 11026-11033

Synthesis and Characterization of Biocompatible, Thermoresponsive ABC and ABA Triblock Copolymer Gelators Chengming Li, Niklaas J. Buurma, Ihtshamul Haq, Colin Turner, and Steven P. Armes* Department of Chemistry, Dainton Building, University of Sheffield, Brook Hill, Sheffield, South Yorkshire, S3 7HF, U.K.

Valeria Castelletto and Ian W. Hamley Department of Chemistry, University of Leeds, Leeds, Yorkshire, LS2 9JT, U.K.

Andrew L. Lewis Biocompatibles UK Ltd., Chapman House, Farnham Business Park, Weydon Lane, Farnham, Surrey, GU9 8QL, U.K. Received June 13, 2005. In Final Form: September 2, 2005 The synthesis of doubly thermoresponsive PPO-PMPC-PNIPAM triblock copolymer gelators by atom transfer radical polymerization using a PPO-based macroinitiator is described. Provided that the PPO block is sufficiently long, dynamic light scattering and differential scanning calorimetry studies confirm the presence of two separate thermal transitions corresponding to micellization and gelation, as expected. However, these ABC-type triblock copolymers proved to be rather inefficient gelators: free-standing gels at 37 °C required a triblock copolymer concentration of around 20 wt%. This gelator performance should be compared with copolymer concentrations of 6-7 wt% required for the PNIPAM-PMPC-PNIPAM triblock copolymers reported previously. Clearly, the separation of micellar self-assembly from gel network formation does not lead to enhanced gelator efficiencies, at least for this particular system. Nevertheless, there are some features of interest in the present study. In particular, close inspection of the viscosity vs temperature plot obtained for a PPO43-PMPC160-PNIPAM81 triblock copolymer revealed a local minimum in viscosity. This is consistent with intramicelle collapse of the outer PNIPAM blocks prior to the development of the intermicelle hydrophobic interactions that are a prerequisite for macroscopic gelation.

Introduction There has been substantial interest in the phase behavior of poly(ethylene oxide)/poly(propylene oxide) [PEO/PPO] block copolymers in aqueous solution, particularly the Pluronic-type triblock systems due to their many commercial applications as emulsifiers, dispersants, and stabilizers.1 Wanka and co-workers determined binary phase diagrams for 12 PEO-PPO-PEO triblock copolymers.2 The phase behavior of many Pluronic-type copolymers has also been studied by Alexandridis and coworkers,3,4 who examined both binary polymer/water mixtures and ternary polymer/water/organic solvent mixtures. Multiple morphologies were observed, including normal and reverse micellar liquid phases, normal and reverse hexagonal-packed cylinder phases, normal and reverse micellar cubic phases, normal and reverse bicontinuous cubic phases, and a lamellar phase. An increase in temperature causes phase boundaries to shift to lower concentration, i.e., the structures swell with water at high temperature as water becomes a less poor solvent for PPO * To whom correspondence should be addressed. E-mail: [email protected]. (1) Hamley, I. W. Block Copolymers in Solution; Wiley: Chichester, 2005. (2) Wanka, G.; Hoffmann, H.; Ulbricht, W. Macromolecules 1994, 27, 4145. (3) Svensson, M.; Alexandridis, P.; Linse, P. Macromolecules 1999, 32, 637. (4) Alexandridis, P. Curr. Opin. Colloid Interface Sci. 1997, 2, 478.

but a worse solvent for PEO. For conventional Pluronics, gelation is due to close packing of micellar aggregates above a certain critical concentration. In contrast, with reverse Pluronic copolymers, the PPO end-blocks can bridge between adjacent micelles, leading to the formation of a physical network.5 Poly(N-isopropylacrylamide) (PNIPAM) exhibits a reversible thermoresponsive phase transition in aqueous solution. 6,7 The lower critical solution temperature (LCST) of PNIPAM is about 32 °C for linear chains in aqueous solution. Thus, PNIPAM dissolves molecularly at ambient temperature but precipitates from hot aqueous solution due to its coil-to-globule transition. This effect is exploited in the preparation of thermosensitive PNIPAM-based hydrogels for biomedical applications such as on/off drug release, the reversible attachment/detachment of cultured cells,8-11 and the surface modification of various substrates.12,13 (5) Mortensen, K.; Brown, W.; Jørgensen, E. Macromolecules 1994, 27, 5654. (6) Heskins, M.; Guilet, J. J. Macromol. Sci. Chem. 1968, A2, 1441. (7) Wu, C.; Wang, X. Phys. Rev. Lett. 1998, 80, 4092. (8) Yoshida, R.; Uchida, K.; Kaneko, Y.; Sakai, K.; Kikuchi, A.; Sakurai, Y.; Okano, T. Nature 1995, 374, 240. (9) Yamada, N.; Okano, T. Makromol. Chem. Rapid Commun. 1990, 11, 571. (10) Qiu, Y.; Park, K. Adv. Drug Delivery Rev. 2001, 53, 321. (11) Canavan, H. E.,; Cheng, X.; Graham, D. J.; Ratner, B. D., Castner, D. G. Langmuir 2005, 21, 1949. (12) Sun, T.; Wang. G.; Fen, L.; Liu. B.; Ma. Y.; Jiang, L.; Zhu, D. Angew. Chem., Int. Ed. 2004, 43, 357.

10.1021/la0515672 CCC: $30.25 © 2005 American Chemical Society Published on Web 10/06/2005

ABC and ABA Triblock Copolymer Gelators

The phosphorylcholine (PC) group is an important motif within cell membranes, and it is now well documented that synthetic PC-based copolymers can be used to produce surface coatings that are remarkably resistant to protein adsorption and bacterial/cellular adhesion.14,15 In particular, biomimetic monomers such as 2-methacryloyloxyethyl phosphorylcholine (MPC) have received increasing attention, since its statistical copolymerization confers clinically proven biocompatibility to a wide range of surface coatings and gels.14c,16 Calorimetric and Raman spectroscopy studies by Ishihara et al. suggest that the excellent biocompatibility of MPC-based coatings is related to the highly hydrophilic nature of the MPC monomer.17 Recently, we showed that MPC can be polymerized with reasonably good control using atom transfer radical polymerization (ATRP).18 This synthetic advance allowed a wide range of new controlled-structure biocompatible diblock copolymers to be prepared, with selected examples showing some potential in the context of pH-sensitive micelles for the delivery of hydrophobic drugs19 and new synthetic vectors for DNA condensation.20 Subsequently, we reported the synthesis of novel, biocompatible pHresponsive gelators based on ABA triblock copolymers, where the central B block comprises MPC and the outer A blocks are composed of 2-(diisopropylamino)ethyl methacrylate (DPA).21 Optimization of the triblock composition allowed us to prepare free-standing gels from 10% triblock copolymer solutions, and preliminary drug release studies were also conducted using dipyridamole as a model hydrophobic drug. Recently we described the synthesis of new thermoresponsive gelators based on PNIPAM-PMPC-PNIPAM triblock copolymers using ATRP.22 These ABA-type triblock copolymers form micellar gels under physiologically relevant conditions, i.e., in phosphate-buffered saline (PBS) solutions at pH 7.4 and 37 °C. The minimum copolymer concentration required for free-standing gels is around 6-7 wt%. Such gels are relatively soft, since they contain more than 90% water. Cell viability studies (13) Hu, Z.; Chen. Y.; Wang. C.; Zheng, Y.; Li, Y. Nature 1998, 393, 149. (14) (a) Hayward, J. A.; Chapman, D. Biomaterials 1984, 5, 135. (b) Durrani, A. A.; Hayward, J. A.; Chapman, D. Biomaterials 1986, 7, 121. (c) Lewis, A. L. Colloids Surf. B: Biointerfaces 2000, 18, 261. (d) Murphy, E. F.; Lu, J. R.; Lewis, A. L.; Brewer, J.; Russell, J.; Stratford, P. Macromolecules 2000, 33, 4345 (15) (a) Ishihara, K. Trends Polym. Sci. 1997, 5, 401. (b) Moro, T.; Takatori, Y.; Ishihara, K.; Konno, T.; Takigawa, Y.; Matsushita, T.; Chung, U.; Nakamura, K.; Kawaguchi, H. Nat. Mater. 2004, 3, 829 (c) Yusa, S.; Fukuda, K.; Yamamoto, T.; Ishihara, K.; Morishima, Y. Biomacromolecules 2005, 6, 663 (16) (a) Driver, M. J.; Jackson, D. J. U.S. Patent No. 5,741,923, 1998. (b) Uchiyama, T.; Watanabe, J.; Ishihara, K. J. Membrane Sci. 2002, 208, 39. (c) Nam, K. W.; Watanabe, J.; Ishihara, K. Biomacromolecules 2002, 3, 100 (17) Iwasaki, Y.; Nakabayashi, N.; Ishihara, K. J. Biomed, Mater. Res. 2001, 57, 72. (18) (a) Lobb, E. J.; Ma, I.; Billingham, N. C.; Armes, S. P.; Lewis, A. L. J. Am. Chem. Soc. 2001, 123, 7913. (b) Ma, I.; Lobb, E, J.; Billingham, N. C.; Armes, S. P.; Lewis, A. L.; Lloyd, A. W.; Salvage, J. P. Macromolecules 2002, 35, 9306. (c) Wang J.; Matyjaszewski, K. J. Am. Chem. Soc. 1995, 117, 5614. (d) Matyjaszewski, K.; Xia, J. Chem. Rev. 2001, 101, 2921. (19) (a) Ma, Y.; Tang, Y.; Billingham, N. C.; Armes, S. P.; Lewis, A. L.; Lloyd, A. W.; Salvage, J. P. Macromolecules 2003, 36, 3475. (b) Rose, S. F.; Salvage, J. P.; West, S. L.; Phillips, G. J.; Hanlon, G. W.; Lloyd, A. W.; Ma, Y.; Armes, S. P.; Billingham, N. C.; Lewis, A. L. J. Controlled Release 2005, 104, 259. (20) (a) Lam, J.; Ma, Y.; Armes, S. P.; Lewis, A. L.; Stolnik, S. J. Controlled Release 2004, 100, 293. (b) Chim, Y. T. A.; Lam, J. K. W.; Ma, Y.; Armes, S. P.; Lewis, A. L.; Roberts, C. J.; Stolnik, S.; Tendler, S. J. B.; Davies, M. C. Langmuir 2005, 21, 3591. (21) (a) Ma, Y.; Tang, Y.; Billingham, N. C.; Armes, S. P. Biomacromolecules 2003, 4, 864. (b) Castelletto, V.; Hamley, I. W.; Ma, Y.; Bories-Azeau, X.; Armes, S. P.; Lewis, A. L. Langmuir 2004, 20, 4306. (22) Li, C.; Tang, Y.; Armes, S. P.; Morris, C. J.; Rose, S. F.; Lloyd, A. W.; Lewis, A. L. Biomacromolecules 2005, 6, 994.

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confirmed that these gels are sufficiently biocompatible to allow V79 hamster lung cells to be cultured in situ, indicating very low or zero cytotoxicities. In the present study, we report the synthesis of a novel doubly thermoresponsive PPO-PMPC-PNIPAM triblock copolymer gelator. This ABC-type copolymer was synthesized by ATRP using a PPO-based macroinitiator to polymerize MPC first, followed by NIPAM (see Figure 1). In principle, the less-hydrophilic PPO block should lead to the formation of noninteracting micelles at temperatures above its LCST, while formation of a micellar gel network was anticipated above the LCST of the PNIPAM block. It was envisaged that separating micellar selfassembly from macroscopic gelation might be more efficient, hence allowing lower copolymer concentrations to be utilized to form gels. The aqueous solution properties of the resulting PPO-PMPC-PNIPAM copolymers were assessed using dynamic light scattering, 1H NMR spectroscopy, high sensitivity differential scanning calorimetry, and rheological measurements. For comparative purposes, we also report some parallel studies concerning PNIPAM-PMPC-PNIPAM triblock copolymer gelators. Experimental Section Materials. MPC monomer (99.5% purity) was kindly donated by Biocompatibles, UK Ltd. N-Isopropylacrylamide (NIPAM, 97%) was purchased from Aldrich and recrystallized from a 3:2 benzene/n-hexane mixture prior to use. Copper(I) bromide (Cu(I)Br 99.999%), 2,2′-bipyridine (bpy, 99%), 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane (Me4Cyclam 98%), and the bifunctional ATRP initiator, diethyl meso-2,5-dibromoadipate (DEDBA), were all purchased from Aldrich and were used without further purification. Two monohydroxy-capped poly(propylene oxide) (PPO-OH) polymers, with mean degrees of polymerization of 33 (Mn ) 1940; Mw/Mn ) 1.09) and 43 (Mn ) 2500; Mw/Mn ) 1.18), respectively, were kindly donated by Cognis Performance Chemicals (Hythe, UK). 2-Bromoisobutyryl bromide (98%) was purchased from Aldrich and used as received. The silica gel 60 (0.063-0.200 mm) was purchased from E. Merck (Darmstadt, Germany) and used as received. Analytical Techniques. 1H NMR spectroscopy. 1H NMR spectra were recorded in either D2O or CD3OD using either a 250 MHz Bruker ACF-250 or a 500 MHz Bruker DRX-500 spectrometer. Aqueous Gel Permeation Chromatography (GPC) Protocols. The number-average molecular weight (Mn) and polydispersity (Mw/Mn) of the MPC homopolymers were assessed by aqueous GPC using two PL Aquagel-OH 40 and Aquagel-OH 30 columns connected to a Polymer Labs ERC-7517A refractive index detector at 20 °C. The eluent was 0.20 M NaNO3 solution containing 50 mM Trizma buffer solution (pH 7.0), and the flow rate was 1.0 mL min-1. The PPO-PMPC diblock copolymer precursors were analyzed by aqueous GPC using essentially the same GPC setup, albeit using a 40:60 v/v methanol/0.20 M NaNO3 mixed aqueous eluent containing 0.01 N NaH2PO4 buffer solution and a flow rate of 0.50 mL min-1. A series of near-monodisperse PEO calibration standards were used for both GPC protocols. Dynamic Light Scattering (DLS) Studies. DLS measurements were conducted on copolymers dissolved in either dilute (0.253.8 g dm-3, depending on the copolymer type) aqueous solution or PBS buffer (pH 7.4) using a Brookhaven model BI-200SM instrument equipped with a 9000AT correlator and a solid-state laser (50 mW, λ ) 532 nm) at a fixed scattering angle of 90°. Rheological Studies. The viscoelastic properties of aqueous solutions of the triblock copolymers were measured using a Rheometrics SR-5000 stress-controlled rheometer. A 40 mm cone with an angle of 2° was used in all measurements. Selected copolymers were dissolved in PBS at pH 7.4 and stored at 5 °C overnight prior to analysis. In the flow measurements, samples were presheared for 3 min at an applied shear stress of 1 Pa. Measurements of the shear storage modulus, G′, and loss modulus, G′′, involved temperature ramps at a frequency of 1

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Figure 1. (a) Reaction scheme for the synthesis of the PPO-PMPC-PNIPAM triblock copolymers via atom transfer radical polymerization (ATRP) using monofunctional PPO-Br ATRP initiator using the macroinitiator route. (b) Schematic representation of the aqueous solution behavior of the PPO-PMPC-PNIPAM triblock copolymers: molecualr dissolution at 5 °C, formation of PPO-core micelles between 10 and 20 °C, and formation of a micellar gel network above 33 °C (which corresponds to the LCST of the outer PNIPAM chains). rad s-1 and dynamic frequency sweeps at a constant stress of 5 Pa at the desired temperature. Differential Scanning Calorimetry (DSC) Studies. The thermal transitions associated with micellization and gelation of aqueous solutions of selected triblock copolymers were studied using a high-precision VP-DSC microcalorimeter (MicroCal, LLC Northampton, MA). The VP-DSC is equipped with two 0.52 mL cells sharing a common headspace sealed with a pressure cap that results in the cells being subjected to an applied pressure of ca. 240 kPa. The aqueous copolymer solution (either 20.0 wt% PPO43-PMPC160-PNIPAM81 or 8.0 wt% PNIPAM89-PMPC250PNIPAM89) and the reference solution (pure water) were each degassed before being loaded slowly into the respective cells at 4 °C. The instrument was operated with the gain mode set to zero, and each solution was alternately heated and cooled at scan rates of 2 and 10 °C per hour. All scans were performed twice and found to be reproducible. Baseline subtractions and further baseline corrections were performed using the instrument software (Microsoft Origin 7.0), yielding the temperaturedependent apparent transition heat capacity (Cp) of copolymer in aqueous solution. Synthesis of Poly(propylene oxide)-Based Macroinitiators. Two PPO-based macroinitiators (PPO33-Br and PPO43-Br, where the subscripts indicate the mean degree of polymerization in each case) were synthesized using a previously reported protocol.23 PPO33-OH (50.0 g, 0.025 mol) or PPO43-OH (50.0 g, 0.019 mol) was dissolved in 250 mL anhydrous toluene. Then, triethylamine (4.21 mL, 0.031 mol for PPO33-OH or 3.90 mL, 0.029 mol for PPO43-OH) was added, and the solution was cooled to 0 °C using an ice bath. 2-Bromoisobutyryl bromide (5.10 mL, 0.031 mol for PPO33-OH or 4.60 mL, 0.029 mol for PPO43-OH) was added dropwise from a dropping funnel over 30 min, and the reaction solution was stirred for a further 48 h at room temperature. The insoluble amine salt was then removed by filtration, and the resulting solution was stirred with activated carbon, dried with MgSO4, filtered again, and dried under (23) Liu, S.; Armes, S. P. J. Am. Chem. Soc. 2001, 123, 9910.

vacuum. Analysis of the 1H NMR spectra of these PPO-OH precursors and the corresponding PPO-Br macroinitiators indicated that the degree of esterification was approximately 100% in each case. Synthesis of the PNIPAM-PMPC-PNIPAM Triblock Copolymers. The synthesis of the PNIPAM89-PMPC250PNIPAM89 triblock copolymer is typical. This copolymer was synthesized in two steps using the so-called ‘macroinitiator’ approach. In the first step, a bifunctional Br-PMPC250-Br macroinitiator was prepared as follows. MPC (3.72 g, 12.5 mmol) was polymerized in 5.0 mL methanol at 20 °C using standard Schlenk techniques, diethyl meso-2,5-dibromoadipate as a bifunctional ATRP initiator (18 mg, 0.05 mmol, target degree of polymerization ) 250) and a Cu(I)Br/2bpy catalyst (14.4 mg, 0.10 mmol Cu(I)Br; 31.2 mg, 0.20 mmol bpy). After 4 h, the MPC conversion was typically more than 98%, as judged by 1H NMR (disappearance of vinyl signals between δ 5.5 and 6.0). The reaction flask was immersed in liquid nitrogen to terminate this first-stage polymerization, excess methanol was added to the frozen solution, and the resulting solution was then passed through a silica gel column to remove the spent ATRP catalyst. After solvent evaporation, the solid polymer was dissolved in deionized water and freeze-dried overnight. The resulting bifunctional Br-PMPC250-Br macroinitiator was obtained as a white powder (3.3 g). The second-stage polymerization to produce the PNIPAM89-PMPC250-PNIPAM89 triblock copolymer was carried out as follows. NIPAM (1.13 g; 10 mmol) and Cu(I)Br/ Me4Cyclam catalyst (7.2 mg, 0.05 mmol Cu(I)Br; 12.8 mg, 0.05 mmol Me4Cyclam) were added to 10.0 mL of degassed methanol in a Schlenk flask immersed in an ice bath to form an homogeneous solution on stirring under a nitrogen atmosphere. The Br-PMPC250-Br macroinitiator (1.84 g; 0.05 mmol bromine) was degassed and added under nitrogen flow; the NIPAM polymerization was allowed to continue for 2 h until 1H NMR analysis indicated no further change in the monomer conversion. The final monomer conversion was calculated using 1H NMR spectroscopy (d4-CD3OD) by comparing the vinyl signals at δ 5.5-6.0 to the single isopropyl proton signal at δ 3.9-4.1. Excess

ABC and ABA Triblock Copolymer Gelators methanol was then added to dilute the reaction solution, which was passed through a silica gel column to remove the spent catalyst. After solvent evaporation, the isolated solid was dissolved in deionized water and any remaining NIPAM monomer was removed by ultrafiltration (the membrane molar mass cutoff was 104 g mol-1) until 1H NMR analysis indicated the absence of any vinyl signals between δ 5.5 and 6.0. Finally, a white powder was obtained by freeze-drying overnight (2.3 g). Synthesis of PPO-PMPC-PNIPAM Triblock Copolymers. The two-step synthesis of the PPO43-PMPC160-PNIPAM81 triblock copolymer is typical. In the first step, a monofunctional PPO43-PMPC160-Br macroinitiator was prepared as follows. MPC (7.10 g, 24 mmol) was polymerized in 17.7 mL of methanol at 20 °C using standard Schlenk techniques, a monofunctional PPO43-Br macroinitiator (398 mg, 0.15 mmol, target degree of polymerization ) 160), and a Cu(I)Br/2bpy catalyst (21.6 mg, 0.15 mmol Cu(I)Br; 46.8 mg, 0.30 mmol bpy). After 24 h, the MPC conversion was typically more than 98%, as judged by 1H NMR (disappearance of vinyl signals between δ 5.5 and 6.0). The reaction flask was immersed in liquid nitrogen to terminate this first-stage polymerization, excess methanol was added to the frozen solution, and the resulting solution was then passed through a silica gel column to remove the spent ATRP catalyst. After solvent evaporation, the solid polymer was dissolved in deionized water and freeze-dried overnight. The resulting monofunctional PPO43-PMPC160-Br (Mn ) 39 200, Mw/Mn ) 1.44; aqueous methanol GPC using vs PEO calibration standards) macroinitiator was obtained as a white powder (6.3 g). The secondstage polymerization to produce the PPO43-MPC160-NIPAM81 triblock copolymer was carried out as follows. NIPAM (1.13 g; 10 mmol) and Cu(I)Br/Me4Cyclam catalyst (14.4 mg, 0.1 mmol Cu(I)Br; 25.6 mg, 0.1 mmol Me4Cyclam) were added to 10 mL degassed methanol in a Schlenk flask immersed in an ice bath and stirred to form a homogeneous solution under a nitrogen atmosphere. The PPO43-PMPC160-Br macroinitiator (5.0 g; 0.10 mmol bromine) was degassed and added under nitrogen flow, and the NIPAM polymerization was allowed to continue for 2 h until 1H NMR analysis indicated no further change in the monomer conversion. The final monomer conversion was calculated using 1H NMR spectroscopy (d4-CD3OD) by comparing the vinyl signals at δ 5.5-6.0 to the single isopropyl proton signal. The reaction solution was then diluted with methanol and passed through a silica gel column to remove the spent catalyst. After solvent evaporation, the isolated solid was dissolved in deionized water and any remaining NIPAM monomer was removed by ultrafiltration (the membrane molar mass cutoff was 104 g mol-1) until 1H NMR analysis indicated the absence of any vinyl signals at δ 5.5-6.0. Finally, the purified PPO43-PMPC160-PNIPAM81 copolymer was obtained as a white powder by freeze-drying overnight (5.4 g). Essentially the same protocol was used to prepare a PPO33-PMPC180-PNIPAM90 triblock copolymer using the PPO33-Br macroinitiator instead of the PPO43-Br macroinitiator. The residual Cu catalyst levels in these two purified triblock copolymers were determined to be approximately 1-2 ppm using inductively coupled plasma atomic emission spectroscopy. The block compositions of the two PPO-PMPCPNIPAM copolymers described in this study were calculated by 1H NMR spectroscopy, using the PPO block as an ‘end-group’ of known mean degree of polymerization (either 33 or 43).

Results and Discussion Background. Recently, we reported22 the synthesis of biocompatible thermoresponsive PNIPAM-PMPCPNIPAM triblock copolymer gelators using the macroinitiator approach. Homopolymerization of MPC using DEDBA as a bifunctional ATRP initiator in conjunction with a Cu(I)Br/2bpy catalyst in methanol at 20 °C was well controlled given the relatively high target degree of polymerization. 1H NMR studies of the reaction solution indicated that the semilogarithmic plot of monomer concentration vs time remained linear up to about 93% conversion. Aqueous GPC studies confirmed the linear evolution of Mn with monomer conversion, with final polydispersities of around 1.30 being achieved. Self-

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blocking experiments conducted with a second shot of MPC monomer suggested that the bifunctional PMPC macroinitiator had an initiator efficiency of close to 100%. However, the ATRP of NIPAM monomer is not as well controlled or as efficient as that of MPC; NIPAM monomer conversions typically reached only 60-90%, but any unreacted monomer can be removed by membrane ultrafiltration using deionized water. In the present study, three well-defined PNIPAM-PMPC-PNIPAM triblock copolymers with approximately fixed PNIPAM block lengths and variable PMPC block lengths were synthesized using the above protocol. The aqueous solution behavior of these three copolymers was compared to that of the doubly thermoresponsive PPO-PMPC-PNIPAM triblock copolymers where appropriate. It is well-known that PPO homopolymer exhibits LCST behavior in aqueous solution.23 Unlike the relatively sharp and molecular weight-independent LCST of 32 °C exhibited by linear PNIPAM, the LCST of PPO is a rather broad, ‘smeared-out’ transition that lies in the 10-20 °C range depending on its degree of polymerization. Since the LCST of PPO is significantly lower than that of PNIPAM, two thermal phase transitions were anticipated for the PPOPMPC-PNIPAM triblock copolymer. Thus, this copolymer was expected to dissolve molecularly in cold water to form well-defined PPO-core micelles at temperatures above the LCST of the PPO block but below the LCST of the PNIPAM block and to form a micellar gel network at temperatures above the LCST of the PNIPAM block (see Figure 1b). One of the fundamental questions to be addressed in this study was whether complete micellar self-assembly prior to formation of the micellar gel network had a beneficial effect on the efficiency of these new PPO-PMPCPNIPAM copolymer gelators. Synthesis of the PPO-PMPC-PNIPAM Triblock Copolymers. The synthesis of two novel PPO-PMPCPNIPAM triblock copolymers was successfully achieved using the macroinitiator approach (see Figure 1a). Unlike low-molecular-weight ATRP initiators, the polymerization rate of MPC using PPO-Br as a macroinitiator in conjunction with the Cu(I)Br/2bpy catalyst in methanol at 20 °C was relatively slow, requiring more than 24 h for an MPC conversion of more than 98%, as judged by 1H NMR studies. Aqueous GPC analysis confirmed that a final polydispersity of around 1.40 was achieved, indicating a reasonably well-controlled polymerization given the relatively high degree of polymerization of 160 that was targeted. Attempts to target higher degrees of polymerization were unsuccessful due to incomplete monomer conversions. The two PPO-MPC-Br macroinitiators were both successfully chain-extended by polymerizing NIPAM under ATRP conditions using a CuBr/Me4Cyclam catalyst22,24a in methanol at 0 °C. However, this second-stage NIPAM polymerization was generally less efficient than the MPC polymerization: 1H NMR studies indicated that NIPAM conversions of around 60-90% were obtained after 3 h, with no further conversions being achieved on longer time scales. Similar problems were reported in our previous work;22 it appears likely that the ATRP of NIPAM has relatively poor living character under these conditions, although our aqueous GPC analyses of these triblock copolymers have not yet proved successful (according to the literature,24 the preferred GPC eluent for PNIPAM is (24) (a) Li, C.; Gunari, N.; Fischer, K.; Janshoff, A.; Schmidt, M. Angew. Chem., Int. Ed. 2004, 43, 1101. (b) Convertine, A. J.; Ayres, N.; Scales, C. W.; Lowe, A. B.; McCormick, C. L. Biomacromolecules 2004, 5, 1177. (c) Schilli, C. M.; Zhang, M.; Rizzardo, E.; Thang, S. H.; Chong, Y. K.; Edwards, K.; Karlsson, G.; Mu¨ller, A. H. E. Macromolecules 2004, 37, 7861

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Table 1. Summary of the Thermoresponsive Gelation Behavior Obtained for the Various MPC-Based ABA and ABC Triblock Copolymers in Aqueous Solution (PBS Buffer, pH 7.4) triblock copolymer architecture and composition

gelation behavior at a given copolymer concentration as judged by tube inversion experiments 5% 10% 15% 20%

PNIPAM81-PMPC200-PNIPAM81 PNIPAM89-PMPC250-PNIPAM89 PNIPAM90-PMPC300-PNIPAM90 PPO33-PMPC180-PNIPAM90

no gel soft gel at 37 °C soft gel at 37 °C no gel

free-standing gel at 37 °C free-standing gel at 37 °C free-standing gel at 37 °C no gel

PPO43-PMPC160-PNIPAM81

no gel

no gel

DMF, whereas PMPC is insoluble in this solvent). Nevertheless, several well-defined PNIPAM-PMPCPNIPAM and PPO-PMPC-PNIPAM copolymers with various block compositions were successfully prepared in which the central MPC block was relatively long compared to the two outer blocks, as described previously for pHresponsive gelators.21 The thermoresponsive behavior of each of these copolymers is summarized in Table 1. DLS, DSC, and NMR Studies of the Triblock Copolymers in Aqueous Solution. Earlier DLS studies on dilute aqueous solutions (0.25 g dm-3) of the PNIPAM89-PMPC250-PNIPAM89 triblock copolymer molecularly dissolved in PBS buffer (pH ) 7.4) had indicated a sharp increase in the scattered light intensity at around 32-33 °C due to the formation of ‘flower’ micelles by the thermoresponsive PNIPAM blocks.22 Macroscopic gelation was observed in more concentrated (6-7 wt%) copolymer solutions, since some fraction of the hydrophobic PNIPAM blocks are involved in intermicelle interactions (with the hydrophilic PMPC block acting as a bridge between adjacent micelles), in addition to their intramicelle interactions. Figure 2 depicts the temperature dependence of the scattered light intensity obtained from DLS studies of dilute aqueous solutions of the two PPO-PMPCPNIPAM triblock copolymers. Two thermal transitions are clearly evident in a 0.05 wt% solution of the PPO43PMPC160-PNIPAM81 triblock copolymer in PBS buffer at pH 7.4, see Figure 2a. There is an initial upturn in scattering intensity above approximately 10 °C that corresponds to the formation of PPO-core micelles and is essentially complete at around 20 °C. A second, moresubstantial increase in light scattering is observed at around 34 °C due to the formation of a micellar gel network (i.e., macroscopic gelation). The three insets depict photographs of a free-flowing, relatively transparent aqueous copolymer solution obtained below 10 °C, a more turbid micellar solution produced between 20 and 34 °C, and a free-standing gel that is formed at around 37 °C. However, in each case, the triblock copolymer concentration is now significantly higher at 20 wt%. In contrast, Figure 2b depicts the DLS data obtained for a 0.38 wt% solution of the PPO33-PMPC180-PNIPAM90 triblock copolymer under the same conditions. In this case, only the higher thermal transition at around 32 °C is observed; there is no evidence for the formation of PPO-core micelles at lower temperatures. This behavior is essentially the same as that reported earlier for a PNIPAM-PMPC-PNIPAM triblock copolymer.22 Moreover, this PPO33-PMPC180-PNIPAM90 triblock copolymer cannot form a free-standing gel at 37 °C even at a copolymer concentration of 20 wt% (gelation does eventually occur on heating this 20 wt% solution up to approximately 56 °C, but unfortunately, this critical gelation temperature is much too high to be useful in the context of potential biomedical applications). Given that these PMPC and PNIPAM block lengths are comparable

free-standing gel at 37 °C free-standing gel at 37 °C free-standing gel at 37 °C no gel at 37 °C; soft gel at 50 °C; free-standing gel at 60°C soft gel at 37 °C; freestanding gel at 40 °C

free-standing gel at 37 °C free-standing gel at 37 °C free-standing gel at 37 °C no gel at 37 °C, soft gel at 46 °C; free-standing gel at 56 °C free-standing gel at 37 °C

to those of the PPO43-PMPC160-PNIPAM81 copolymer, this reduced gelation efficiency suggests that there is a minimum PPO block length required for the formation of well-defined micelles at lower temperature: a mean degree

Figure 2. (a) Temperature dependence of the scattered light intensity for a 0.05 wt% PPO43-PMPC160-PNIPAM81 triblock copolymer solution in PBS buffer at pH 7.4. Inset shows three digital photographs recorded for the same copolymer dissolved at a much higher concentration (20 wt%) in PBS buffer: (left) a transparent free-flowing copolymer solution at 5 °C, (middle) a slightly turbid free-flowing micellar solution at 20 °C, and (right) the free-standing micellar gel formed at 37 °C. (b) Temperature dependence of the scattered light intensity obtained for a 0.38 wt% PPO33-PMPC180-PNIPAM90 triblock copolymer solution in PBS buffer solution at pH 7.4.

of polymerization of only 33 is simply not sufficient to ensure significant hydrophobic interactions between the PPO chains.25 This hypothesis is confirmed by DLS studies of dilute aqueous solutions of these two PPO-PMPC(25) The water-solubility of PPO homopolymer depends on its mean degree of polymerization (Dp). At a mean Dp of 33 or lower, PPO is water-soluble, although it exhibits inverse temperature solubility behavior above ambient temperature. However, at a mean Dp of 43, PPO is water-insoluble. However, it is well known that the water solubility of PPO is also influenced by end-group effects. Given the highly hydrophilic nature of the PMPC block, it is not unreasonable that the PPO33-based triblock copolymer exhibits poor gelation efficiency, but the PPO43-based triblock has reasonable gelation efficiency.

ABC and ABA Triblock Copolymer Gelators

PNIPAM copolymers, see Figure 2. In summary, the copolymer gelation behavior was relatively sensitive to the degree of polymerization of the PPO block. In the rest of this paper we focus on the aqueous solution properties of the PPO43-PMPC160-PNIPAM81 triblock gelator, making comparisons where appropriate with the PNIPAM-PMPC-PNIPAM triblock copolymers previously reported. It is emphasized that each of these micellar gels are purely physical in nature, since they all revert to freeflowing solutions below their critical gelation temperatures. The aqueous gelation behavior of the various triblock copolymers is summarized in Table 1. For ABA triblock copolymers of PNIPAM-PMPC-PNIPAM, the gelation efficiency was relatively high. The minimum concentrations for the formation of a free-standing gel in aqueous or PBS buffer (pH 7.4) solutions were 8.2%, 6.5%, and 7.2% for the PNIPAM81-PMPC200-PNIPAM81, PNIPAM89-PMPC250-PNIPAM89, and PNIPAM81PMPC300-PNIPAM90 copolymers, respectively. Moreover, soft gels can be formed below 5 wt%. For PPO-PMPCPNIPAM-type triblock copolymers, the gelation efficiency was relatively low. Although micellar aggregates were formed prior to gelation in both aqueous and PBS solutions, the minimum copolymer concentrations required for the formation of free-standing gels were relatively high under all conditions. For example, a 20 wt% solution of the PPO33-PMPC180-PNIPAM90 copolymer had to be heated to 56 °C to produce a gel, while the minimum copolymer concentrations required for gelation of the PPO43PMPC180-PNIPAM81 copolymer were 15 wt% at 40 °C and 18.5 wt% at 37 °C, respectively. High-sensitivity DSC traces obtained for aqueous copolymer solutions comprising 8% PNIPAM89-PMPC250PNIPAM89 and 20% PPO43-PMPC160-PNIPAM81 are depicted in Figure 3. The latter copolymer solution is characterized by two features at approximately 15 and 32 °C (see Figure 3a), which correlate very closely with the temperature-dependent light scattering studies shown in Figure 2a. These two thermal transitions correspond to micellization and macroscopic gelation, respectively. The peak associated with micellization is broad and weak, as expected for PPO chains, while the peak assigned to the PNIPAM chains is sharp and intense, as expected for a coil-to-globule transition. In contrast, only the highertemperature feature is observed for the PNIPAM89PMPC250-PNIPAM89 copolymer solution (see Figure 3b), since this copolymer does not contain any PPO chains. It is noteworthy that this thermal transition, which is associated with simultaneous micellization and gelation, is shifted to around 35 °C, compared to the LCST of 32 °C that is usually observed for PNIPAM. The effect of reversing the temperature ramp was also examined in these DSC studies, and some hysteresis effects were evident. In the case of the PNIPAM-PMPC-PNIPAM triblock gelator, a distinctive shoulder appeared in the thermal transition during the cooling ramp that was not observed in the heating ramp. Similar observations have been recently reported by Ding et al. for aqueous solutions of linear PNIPAM homopolymer.26 According to these workers, this shoulder suggests that additional hydrogen bonding between the PNIPAM chains may be formed in their collapsed state. This effect was not apparent in the DSC traces obtained for the PPO-PMPC-PNIPAM copolymer solutions, although the weaker feature assigned to the micellization of the PPO chains was shifted to lower (26) Ding, Y.; Ye, X.; Zhang, G. Macromolecules 2005, 38, 904.

Langmuir, Vol. 21, No. 24, 2005 11031

Figure 3. Temperature dependence of the apparent heat capacity (Cp) for (a) 20% aqueous solution of the PPO43PMPC160-PNIPAM81 triblock copolymer at pH 7.0 and (b) 8% aqueous solution of the PNIPAM89-PMPC250-PNIPAM89 triblock copolymer at pH 7.0. Both the heating (dash line) and cooling (solid line) rates were 2.0 °C min-1 for each copolymer solution. Moreover, similar results were obtained with heating and cooling rates of 10.0 °C min-1 for each copolymer solution.

Figure 4. Variable-temperature 1H NMR spectra (D2O) recorded for a 20% aqueous solution of the PPO43-MPC160NIPAM81 triblock copolymer.

temperature and was somewhat attenuated during the cooling ramp. The gelation behavior of the PPO43-PMPC160PNIPAM81 copolymer was examined both as a free-flowing solution at or below ambient temperature and also as a free-standing gel at higher temperatures using 1H NMR spectroscopy. Typical spectra are depicted in Figure 4 for a 20 wt% copolymer solution in D2O. All of the NMR signals expected for the central PMPC block and the PPO and PNIPAM outer blocks are visible at 5 °C (lower spectrum), indicating a high degree of solvation and mobility for all three components at this temperature. The signal ‘c’ at 1.2 ppm due to the methyl groups on the PPO block (partially overlapping with the methacrylate signal ‘d’) is somewhat reduced at 20 °C (middle spectrum), indicating

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Figure 5. Temperature dependence of the solution viscosity at a constant stress of 5 Pa for a PPO43-PMPC160-PNIPAM81 triblock copolymer dissolved in PBS buffer at 10 (9), 15 (2), and 20 wt% (b). The inset shows the same 20 wt% copolymer solution analyzed more closely over a narrower temperature range.

significant reduction in solvation and mobility for the PPO chains at this temperature. This is consistent with the formation of PPO-core micelles, as indicated by the DLS studies. On increasing the solution temperature up to 37 °C (upper spectrum), the two NMR signals, ‘b’ and ‘a’, due to the PNIPAM blocks are no longer visible, indicating that the PNIPAM chains also become hydrophobic at this temperature. This is consistent with the formation of a micellar gel network based on hydrophobic associations between the PNIPAM chains on adjacent micelles. Rheological Behavior of Triblock Copolymer Gelators in Aqueous Solution. Rheological studies were conducted on the doubly responsive PPO43-PMPC160PNIPAM81 and also the three PNIPAM-PMPC-PNIPAM triblock copolymers. A typical viscosity vs temperature plot of an 8.0 wt% PNIPAM89-PMPC250-PNIPAM89 aqueous copolymer solution obtained at a shear stress of 1.0 Pa was reported previously.22 At 20 °C, this copolymer solution behaved as a Newtonian fluid, and below 30 °C, its viscosity was less than 0.1 Pa‚s. A sharp increase in viscosity occurred at around 35 °C, indicating the onset of gelation and at 37 °C the viscosity of the gel exceeded 100 Pa‚s. It is also noteworthy that the viscosity increased monotonically for this PNIPAM89-PMPC250-PNIPAM89 copolymer over the temperature range studied (20-50 °C) As a comparison, typical viscosity vs temperature plots obtained for 10, 15, and 20 wt% aqueous solutions of the PPO43-PMPC160-PNIPAM81 copolymer are shown in Figure 5. At copolymer concentrations of 10 and 15 wt%, the solution viscosity remained relatively low over the 5-55 °C range, indicating typical Newtonian fluid behavior and no gelation under these conditions. However, the behavior of the 20 wt% copolymer solution was different. In this case, the solution viscosity was much more strongly temperature-dependent, with free-standing gels being formed at approximately 37 °C. Moreover, there was some evidence for a kink in the viscosity data at around 32 °C. To examine this unusual feature, these viscosity measurements were repeated in more detail over a much narrower temperature range (see inset in Figure 5). It is clear that the solution viscosity exhibits a local minimum at approximately 33 °C. It is emphasized that no such feature is observed in any of the viscosity vs temperature plots obtained for the PNIPAM-PMPC-PNIPAM triblock copolymer gelators. A tentative explanation for this unexpected effect is as follows. The PPO43-PMPC160PNIPAM81 copolymer exists as PPO-core micelles at temperatures below 32 °C, with the hydrophilic PNIPAM

Li et al.

Figure 6. Temperature dependence of the storage modulus (G′) (b) and loss modulus (G′′) (O) for a 20 wt% PPO43PMPC160-PNIPAM81 triblock copolymer dissolved in PBS solution at pH 7.4. Note the inflections in both sets of data that occur at around 33 °C.

Figure 7. Frequency dependence of the storage modulus (G′) and loss modulus (G′′) at a constant applied stress of 5 Pa for (a) a 9 wt% PNIPAM81-PMPC200-PNIPAM81 triblock copolymer dissolved in PBS buffer at 35 (G′, b; G′′, O) and 40 °C (G′, 9; G′′, 0), respectively, and (b) a 20 wt% PPO43-PMPC160PNIPAM81 triblock copolymer dissolved in PBS buffer at 10 (G′, b; G′′, O), 25 (G′, 9; G′′, 0), and 37 °C (G′, 2; G′′, 4), respectively.

chains being located on the periphery of these micelles. At around 33 °C, which is only slightly higher than the LCST of linear PNIPAM homopolymer, the PNIPAM chains undergo a coil-to-globule transition7 and become relatively hydrophobic. However, this rapid intramicelle collapse occurs prior to the development of significant attractive intermicelle interactions. At slightly higher temperatures the PNIPAM chains on neighboring micelles begin to interact, leading to the formation of a micellar gel network. This is a different situation to that encountered with the PNIPAM-PMPC-PNIPAM triblock copolymers, where micellization and gelation occur simul-

ABC and ABA Triblock Copolymer Gelators

Langmuir, Vol. 21, No. 24, 2005 11033

PNIPAM81 and the PPO43-PMPC160-PNIPAM81 triblock copolymers. This means that these gels are relatively weak, which is unsurprising given their relatively high water contents (80-90%).

taneously. Thus, the unusual feature observed in Figure 5 is a direct consequence of the doubly responsive nature of the novel PPO-PMPC-PNIPAM gelator. Studies of the temperature dependence of the storage modulus (G′) and loss modulus (G′′) for a 20 wt% aqueous solution of PPO43-MPC160-NIPAM81 are shown in Figure 6. G′ is lower than G′′ for all temperatures below 33 °C, which is typical behavior for a Newtonian fluid. The critical gelation temperature is where G′ is equal to G′′ and is approximately 33 °C. Above this gel point, G′ is greater than G′′, which is characteristic of an elastic gel.27 Moreover, it is apparent that both G′ and G′′ decrease at around the same characteristic temperature at which the local minimum in solution viscosity was observed (see Figure 6). The shear rate dependence of G′ and G′′ at a constant shear stress of 5 Pa is shown in Figure 7a for a 9% PNIPAM81-PMPC200-PNIPAM81 copolymer solution at various temperatures. G′ is always less than G′′ for temperatures below 35 °C, indicating Newtonian fluid behavior prior to gelation. In contrast, G′ exceeds G′′ at 40 °C, indicating that gel formation occurred at this temperature (the actual critical gelation temperature for this copolymer solution was approximately 37 °C). In Figure 7b, the shear rate dependence of G′ and G′′ at a constant shear stress of 5 Pa is shown for a 20% aqueous solution of the doubly responsive PPO43PMPC160-PNIPAM81 copolymer. G′ is lower than G′′ at 10 and 25 °C, indicating typical Newtonian fluid behavior at these temperatures, regardless of whether micellization had occurred or not. In contrast, G′ exceeded G′′ at 37 °C, indicating gel formation at this physiologically relevant temperature. However, the magnitudes of both G′ and G′′ are relatively low for both the PNIPAM81-PMPC200-

New doubly thermoresponsive PPO-PMPC-PNIPAM triblock copolymer gelators have been successfully synthesized by ATRP using a PPO-based macroinitiator. Provided that the PPO block is sufficiently long, two thermal transitions corresponding to micellization and gelation are observed in DLS and DSC studies, as expected. However, the separation of micellar self-assembly from gel network formation unfortunately did not lead to enhanced gelator efficiencies: these ABC-type triblock copolymers proved to be somewhat less effective gelators than the PNIPAM-PMPC-PNIPAM triblock copolymers reported earlier. Nevertheless, there are some interesting aspects in the present study. In particular, close inspection of the viscosity vs temperature plot obtained for a PPO43PMPC160-PNIPAM81 triblock copolymer revealed a local minimum in viscosity, which is consistent with intramicelle collapse of the outer PNIPAM blocks prior to the development of the intermicelle hydrophobic interactions that are a prerequisite for macroscopic gelation.

(27) Berlinova, I. V.; Dimitrov, I. V.; Vladimirov, N. G.; Samichkov, V.; Ivanov, Y. Polymer 2001, 42, 5963.

LA0515672

Conclusions

Acknowledgment. S.P.A. thanks EPSRC (GR/R29260) and Biocompatibles for postdoctoral support for C.L. S.P.A. is a recipient of a Royal Society/Wolfson Research Merit Award. Biocompatibles is also thanked for supplying the MPC monomer and for permission to publish this work. We also wish to thank a reviewer for their constructive criticism of this manuscript.