Synthesis and Characterization of Biocompatible Thermo-Responsive

Biomacromolecules 2005, 6, 994-999

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Synthesis and Characterization of Biocompatible Thermo-Responsive Gelators Based on ABA Triblock Copolymers Chengming Li, Yiqing Tang, and Steven P. Armes* Department of Chemistry, University of Sheffield, Brook Hill, Sheffield, South Yorkshire S3 7HF, United Kingdom

Christopher J. Morris, Susanna F. Rose, and Andrew W. Lloyd School of Pharmacy and Biomolecular Sciences, University of Brighton, Moulsecoomb, Brighton, East Sussex BN2 4GJ, United Kingdom

Andrew L. Lewis Biocompatibles UK Ltd., Chapman House, Farnham Business Park, Weydon Lane, Farnham, Surrey GU9 8QL, United Kingdom Received October 22, 2004; Revised Manuscript Received December 3, 2004

The synthesis of biocompatible, thermo-responsive ABA triblock copolymers in which the outer A blocks comprise poly(N-isopropylacrylamide) and the central B block is poly(2-methacryloyloxyethyl phosphorylcholine) is achieved using atom transfer radical polymerization with a commercially available bifunctional initiator. These novel triblock copolymers are water-soluble in dilute aqueous solution at 20 °C and pH 7.4 but form free-standing physical gels at 37 °C due to hydrophobic interactions between the poly(Nisopropylacrylamide) blocks. This gelation is reversible, and the gels are believed to contain nanosized micellar domains; this suggests possible applications in drug delivery and tissue engineering. Introduction In recent years stimulus-responsive hydrogels that are sensitive to environmental stimuli (such as temperature, solution pH, or salt concentration) have attracted considerable attention because of their potential applications in drug delivery and tissue engineering.1-7 The phosphorylcholine motif is an important component of cell membranes; hence, synthetic copolymers containing so-called “bio-inspired” monomers such as 2-methacryloyloxyethyl phosphorylcholine [MPC] can be used to produce highly biocompatible surface coatings that are remarkably resistant to protein adsorption and bacterial/cellular adhesion.8-10 Moreover, it is well-known that linear poly(N-isopropylacrylamide) [PNIPAM] exhibits a reversible phase transition in aqueous solution at around 32 °C, with precipitation occurring above this temperature.11,12 Thermo-responsive lightly cross-linked PNIPAM-based microgels have been studied for their on/ off drug release and reversible attachment/detachment of cultured cells.13,14 However, as far as we are aware, there are no reports of any copolymers that contain both MPC and N-isopropylacrylamide (NIPAM). In earlier work, we described the synthesis of pHresponsiVe ABA triblock copolymer gelators based on MPC and 2-(diisopropylamino)ethyl methacrylate [DPA].15 Herein we report the synthesis and characterization of three new * To whom correspondence should be addressed. Tel.: UK + 114-2229342. E-mail: [email protected].

types of thermo-responsiVe ABA triblock copolymers where MPC is the central B block and the outer A blocks are based on either 2-(dimethylamino)ethyl methacrylate (DMA), 2hydroxyethyl methacrylate (HEMA), or NIPAM (see Figure 1). Preliminary results for the gelation behavior of the NIPAM-based ABA triblock copolymers are presented. Experimental Section Materials. MPC monomer (99.9% purity) was kindly donated by Biocompatibles, U.K. 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%), 1,4,8,11tetramethyl-1,4,8,11-tetraazacyclo-tetradecane (Me4Cyclam, 98%), and the bifunctional atom transfer radical polymerization (ATRP) initiator, diethyl-meso-2,5-dibromoadipate (DEDBA) were all purchased from Aldrich and were used without further purification. Synthesis of DMA90-MPC250-DMA90 and HEMA90MPC250-HEMA90 ABA Triblock Copolymers. A typical synthesis of a DMA90-MPC250-DMA90 and HEMA90MPC250-HEMA90 triblock copolymer was carried out using the bifunctional DEDBA initiator and the CuBr/2bpy catalyst in methanol at 20 °C via sequential monomer addition. MPC (3.72 g, 12.5 mmol) was polymerized in 5 mL of methanol at 20 °C using standard Schlenk techniques, diethyl-meso2,5-dibromoadipate as a bifunctional ATRP initiator (18 mg,

10.1021/bm049331k CCC: $30.25 © 2005 American Chemical Society Published on Web 01/11/2005

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Figure 1. Reaction scheme for the synthesis of the NIPAM-MPC-NIPAM triblock copolymers via ATRP using a commercially available bifunctional ATRP initiator using the macro-initiator route. Similar triblock copolymers were also synthesized in “one-pot” syntheses via sequential monomer addition where the NIPAM monomer was replaced with either DMA or HEMA.

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). Degassed DMA monomer (1.41 g, 9 mmol, target degree of polymerzation ) 90 for each block) in 1.5 mL of methanol or HEMA (1.17 g, 9 mmol, target degree of polymerization ) 90 for each block) in 1.3 mL of methanol was injected by syringe. After 24 h the second monomer conversion was typically more than 98% as judged by 1H NMR (disappearance of vinyl signals between δ 5.5 and 6.0). Excess methanol was added to the solution, and the resulting solution was then passed through a silica gel column [silica gel 60 (0.063-0.200 mm)] to remove the spent ATRP catalyst. After solvent evaporation, the solid polymer was dissolved in deionized water and freeze-dried overnight. The resulting DMA90-MPC250DMA90 or HEMA90-MPC250-HEMA90 triblock copolymer was obtained as a white powder (4.0 or 3.7 g, respectively), Synthesis of MPC125-NIPAM89 BA Diblock Copolymer. A typical synthesis of a MPC125-NIPAM89 diblock copolymer was carried out in two steps using the so-called “macro-initiator” approach. In the first step, a monofunctional MPC125-Br macro-initiator was prepared as follows. MPC (7.4 g, 25 mmol) was polymerized in 10 mL of methanol at 20 °C using standard Schlenk techniques, monofunctional ME-Br initiator [56 mg, 0.2 mmol, target degree of polymerization ) 125; synthesized by reacting 4-(2-hydroxyethyl)morpholine (10.0 g, 0.0763 mol) with stoichiometric quantities of 2-bromoisobutyryl bromide (17.549 g, 0.0763 mol) and triethylamine (7.706 g, 0.0763 mol) in dry toluene (200 mL) for 48 h at ambient temperature, as described20 previously], and a Cu(I)Br/2bpy catalyst [28.8 mg, 0.20 mmol Cu(I)Br; 62.4 mg, 0.40 mmol bpy]. After 4.5 h, the MPC conversion was typically more than 98% as judged by 1 H 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 though a silica gel column [silica gel 60 (0.0630.200 mm)] to remove the spent ATRP catalyst. After solvent evaporation, the solid polymer was dissolved in deionized water and freeze-dried overnight. The resulting monofunctional MPC125-Br macro-initiator was obtained as a white powder (7.0 g). The second-stage polymerization to produce the MPC125-NIPAM89 diblock copolymer was carried out as follows. NIPAM (1.7 g, 15 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 15 mL of degassed methanol in a Schlenk flask and stirred in an ice bath to form a homogeneous solution under a nitrogen atmosphere. The MPC125-Br macro-initiator (3.7 g; 0.1 mmol bromine) was degassed and added under a nitrogen atmosphere, and the NIPAM polymerization was allowed to continue for 2 h until 1H NMR analysis indicated no further change in conversion. The monomer conversion was calculated by comparing the vinyl signals between δ 5.5-6.0 to the single isopropyl proton signal using 1H NMR spectroscopy (d4CD3OD). Excess 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 (membrane molar mass cutoff ) 3 × 103 g mol-1) until 1H NMR analysis indicated the absence of any vinyl signals between δ 5.5 and δ 6.0. Finally, the white diblock copolymer was obtained by freeze-drying overnight (4.2 g). Synthesis of NIPAMn-MPCm-NIPAMn ABA Triblock Copolymer. A typical synthesis of a NIPAM89-MPC250NIPAM89 triblock copolymer was carried out in two steps using the so-called “macro-initiator” approach. In the first step, a bifunctional Br-MPC250-Br macro-initiator was prepared as follows. MPC (3.72 g, 12.5 mmol) was polymerized in 5 mL of methanol at 20 °C using standard Schlenk techniques, DEDBA 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

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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 δ 6.0). The reaction flask was immersed in liquid nitrogen to terminate this firststage polymerization, excess methanol was added to the frozen solution, and the resulting solution was then passed though a silica gel column [silica gel 60 (0.063-0.200 mm)] to remove the spent ATRP catalyst. After solvent evaporation, the solid polymer was dissolved in deionized water and freeze-dried overnight. The resulting bifunctional BrMPC250-Br macro-initiator was obtained as a white powder (3.3 g). The second-stage polymerization to produce the NIPAM89-MPC250-NIPAM89 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 mL of degassed methanol in a Schlenk flask and stirred in an ice bath to form a homogeneous solution under a nitrogen atmosphere. The Br-MPC250-Br macro-initiator (1.84 g; 0.05 mmol bromine) was degassed and added under a nitrogen atmosphere, and the NIPAM polymerization was allowed to continue for 2 h until 1H NMR analysis indicated no further change in conversion. The monomer conversion was calculated by comparing the vinyl signals between δ 5.5-6.0 to the single isopropyl proton signal using 1H NMR spectroscopy (d4-CD3OD). Excess 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 (membrane molar mass cutoff ) 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). Copolymer Characterization. 1H NMR spectra were recorded in either D2O or CD3OD using either a 300 MHz Bruker Avance DPX300 or a 500 MHz Bruker AMX500 spectrometer. The number-average molecular weights (Mn) and molecular weight distributions of the MPC homopolymers were assessed by aqueous gel permeation chromatography (GPC) using a Pharmacia Biotech “Superose 6” column connected to a Polymer Labs ERC-7517A refractive index detector at 20 °C. The eluent was a 0.2 M NaNO3 solution containing a 50 mM Trizma buffer solution, and the flow rate was 1.0 mL min-1. A series of nearmonodisperse poly(ethylene oxide) standards were used for calibration. Dynamic light scattering (DLS) studies were performed in aqueous solutions using a Brookhaven model BI-200SM equipped with a correlator 9000AT and a solidstate laser (50 mW, λ ) 532 nm) using a scattering angle of 90°. Gel Rheology Studies. The rheological properties of the synthesized ABA triblock copolymers in phosphate buffer solution (PBS) were assessed using a Carri-Med CSL2 100 rheometer (TA Instruments). A 6-cm cone with an angle of 2° was selected, and precautions were taken to avoid water vaporation from the gels during these flow measurements. The copolymers were dissolved in the buffer at the desired concentration and stored at 0 °C overnight prior to gelation

Li et al.

Figure 2. Homopolymerization of MPC in methanol via ATRP at 20 °C: (a) conversion versus time and semilogarithmic plot of monomer concentration versus time [conditions: MPC (3.72 g, 125 mmol); 5 mL of methanol; target DPn ) 250; relative molar ratios were [DEDBA]/[Cu(I)]/[L] ) 1:2:4]; (b) evolution of molecular weight versus conversion obtained under the conditions stated in part a.

studies. Copolymer gels or solutions were pre-sheared for 3 min at a constant applied shear stress of 1.00 Pa before flow behavior was examined. The heating rate was typically 3 °C min-1. Cell Viability Studies. Five thousand V79 hamster lung cells were seeded into two well chamber slides containing 5, 10, and 20% NIPAM81-MPC200-NIPAM81 copolymer solutions in cell culture media (Dulbecco’s modified essential media + 10% fetal calf serum). These solutions were then gelled at 37 °C and incubated for 24, 48, or 72 h in a 5% CO2 atmosphere prior to examination of the gels using light microscopy. Because live/dead staining of cells proved problematic due to dye adsorption onto the polymer gels, cell viability was confirmed by removing the cells from the gels and culturing for a further 48 h on tissue culture plastic prior to examination. Results and Discussion We previously reported the use of DEDBA as a bifunctional ATRP initiator in combination with a CuBr/bpy catalyst in methanol at 20 °C to produce MPC-based ABA triblock copolymers. 15 However, the detailed polymerization kinetics using this bifunctional ATRP initiator has not yet been reported. The results (see Figure 2) indicate reasonable “living” character. The semilogarithmic plot of the monomer concentration versus polymerization time was linear up about 93% conversion as judged by 1H NMR, and aqueous GPC studies confirmed that there is a linear evolution in Mn with monomer conversion, with final polydispersities of around 1.30 being achieved.

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Table 1. Summary of the Gelation Behavior Obtained for the Various MPC-Based ABA Triblock Copolymers in PBS Solutions at 37 °C gelation behavior at given copolymer concentration ABA triblock copolymer composition

5%

10%

15%

20%

DMA90-MPC250-DMA90 HEMA90-MPC250-HEMA90 MPC125-NIPAM89 NIPAM81-MPC200-NIPAM81 NIPAM89-MPC250-NIPAM89 NIPAM90-MPC300-NIPAM90

no no no no soft gel soft gel

no no no free-standing gelb free-standing gelc free-standing geld

no no no free-standing gel free-standing gel free-standing gel

noa noa noa free-standing gel free-standing gel free-standing gel

a No gels were formed at 20% copolymer concentration, even at 60 °C. gels at 37 °C in PBS were 8.3, 6.5 and 7.2%, respectively.

Figure 3. Aqueous GPC curves obtained for a chain-extended MPC400 homopolymer (Mn ) 69 400, Mw/Mn ) 1.50) prepared using a purified bifunctional Br-MPC200-Br macro-initiator (Mn ) 29 600, Mw/Mn ) 1.37) in methanol at 20 °C. Relative molar ratios were [MPC]/ [Br-MPC200-Br]/[Cu(I)]/[bpy] ) 200:1:2:4; total target DPn ) 400.

The syntheses of the target DMA-MPC-DMA and HEMA-MPC-HEMA triblock copolymers were readily achieved using the bifunctional DEDBA initiator and the CuBr/2bpy catalyst in methanol at 20 °C via sequential monomer addition. In the case of the DMA-MPC-DMA triblock, aqueous GPC measurements [vs poly(2-vinylpyridine) calibration standards] indicated reasonably low polydispersities for both the MPC macro-initiator (Mw/Mn ) 1.3-1.5) and the final triblock copolymer (Mw/Mn ) 1.51.6). However, no GPC measurements were possible for the HEMA-MPC-HEMA triblock copolymer because we were unable to find a suitable GPC eluent. Unfortunately, 20% aqueous solutions of DMA90-MPC250-DMA90 and HEMA90-MPC250-HEMA90 in PBS did not form gels even at temperatures as high as 60 °C. This is believed to be due to the relatively weak hydrophobic interactions between the DMA (or HEMA) chains. In view of this, we elected to synthesize NIPAM-MPC-NIPAM triblock copolymers, because PNIPAM is relatively hydrophobic (compared to most other water-soluble polymers) at temperatures above its characteristic cloud point of 32 °C. However, the syntheses of the target NIPAMn-MPCmNIPAMn triblock copolymers were somewhat problematic because the ATRP of these two monomers requires different ligands and polymerization temperatures for optimal “living” character.16,17 Despite this known incompatibility, one-pot syntheses via sequential monomer addition were attempted, but without success. For example, the ATRP of MPC was well controlled using the DEDBA initiator and the CuBr/ 2bpy catalyst in methanol at 20 °C but almost no secondstage polymerization occurred on addition of NIPAM in situ

b -dThe

minimum copolymer concentrations required to produce free-standing

Figure 4. Temperature dependence of the scattered light intensity for a NIPAM89-MPC250-NIPAM89 triblock copolymer at 0.25 g dm-3 in PBS. Inset shows two digital photographs of (left) the free-flowing PBS at 20 °C and (right) the free-standing physical gel formed at 37 °C by a 6.5% NIPAM89-MPC250-NIPAM89 copolymer in PBS.

under these conditions. It was known that NIPAM could be polymerized using CuBr/Me4Cyclam catalyst in organic solvents at low temperature.16 Our attempt to promote the in situ polymerization of NIPAM by addition of the preferred CuBr/Me4Cyclam catalyst was also unsuccessful. Thus, we were forced to adopt a two-step strategy that involved isolation and purification of the MPC-based macro-initiators. First “self-blocking” control experiments were conducted by adding MPC to a purified bifunctional Br-MPC200-Br macro-initiator in the presence of a CuBr/bpy catalyst in methanol at 20 °C. The results (see Figure 3) confirmed that chain extension occurred as expected and that the overall MPC conversion was almost 100% as judged by 1H NMR. GPC analysis indicated that, within experimental error, the expected twofold increase in Mn had occurred. Having demonstrated its activity, the Br-MPC200-Br macro-initiator was then used to polymerize NIPAM in methanol at 0 °C with the preferred CuBr/Me4Cyclam catalyst. This second-stage NIPAM polymerization was usually less efficient than the MPC polymerization. Nevertheless, 1H NMR studies indicated that NIPAM conversions of 65-90% were achieved after around 2 h. Any unreacted NIPAM monomer was removed completely (as judged by 1 H NMR) by membrane ultrafiltration using deionized water. Similar results were reported previously by Li et al.16 The residual Cu catalyst levels in the triblock copolymers were determined to be approximately 1-2 ppm using inductively coupled plasma atomic emission spectroscopy, as described previously.17 Various well-defined ABA triblock copolymers

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Figure 5. Variable-temperature 1H NMR spectra recorded for a 6.5% aqueous solution of the NIPAM89-MPC250-NIPAM89 triblock copolymer in D2O.

Figure 6. Viscosity versus temperature plot obtained for an 8% aqueous solution of the NIPAM89-MPC250-NIPAM89 triblock copolymer in PBS.

with different block compositions were prepared in which the central MPC block was relatively long compared to the two outer blocks, as described previously for pH-responsive gelators.15 The thermo-responsive behavior of each of these copolymers is summarized in Table 1. Unfortunately, aqueous GPC analyses of these NIPAMn-MPCm-NIPAMn triblock copolymers have not yet proved successful. Nevertheless, these NIPAMn-MPCm-NIPAMn triblock copolymers can be molecularly dissolved in either pure water or a PBS (pH 7.4) at 20 °C. The PNIPAM blocks become hydrophobic above approximately 37 °C, leading to attractive interchain interactions. This phase transition is slightly higher than the cloud point of 32 °C usually exhibited by PNIPAM

Li et al.

homopolymer and probably reflects the influence of the highly hydrophilic MPC block. Similar effects have been reported previously.18 DLS studies on dilute aqueous solutions (0.25 g dm-3) of the NIPAM89-MPC250-NIPAM89 triblock copolymer dissolved in PBS buffer indicated a sharp increase in the scattered light intensity at around 32-33 °C (see Figure 4) due to the onset of micelle formation by the thermo-responsive NIPAM blocks. At sufficiently high copolymer concentrations (above 6.58.3%, depending on the copolymer composition), freestanding physical gels were produced at 37 °C due to the hydrophobic interchain interactions of NIPAM blocks. This is illustrated in the inset in Figure 4, which shows digital photographs of two sample tubes containing the same 6.5 wt % NIPAM89-MPC250-NIPAM89 triblock copolymer solution in PBS buffer at 20 and 37 °C, respectively. These macroscopic gels are purely physical in nature, because they revert to free-flowing solutions below 37 °C. It is worth emphasizing that a relatively concentrated (20%) aqueous solution of MPC125-NIPAM90 diblock copolymer (prepared using a monofunctional ATRP initiator; see Experimental Section) in PBS buffer does not form free-standing gels even at 60 °C. This control experiment confirms that triblock architectures are essential for gelation, because the second NIPAM block is necessary to ensure that intermicelle bridging occurs.15 The NIPAM89-MPC250-NIPAM89 triblock copolymer was dissolved in D2O at 6.5 wt % and was examined as both a free-flowing solution and a free-standing gel using variabletemperature 1H NMR spectroscopy (see Figure 5). At 20 and 33 °C the expected NIPAM signals are observed, indicating a high degree of solvation and mobility for these blocks under these conditions. Moreover, the apparent block composition at 33 °C is essentially the same as that at 20 °C, which suggests little or no contamination by PNIPAM homopolymer (because this impurity, if present, is expected to precipitate above 32 °C). However, the PNIPAM signals are no longer visible at 37 °C, indicating that the PNIPAM blocks become hydrophobic at this temperature. Small-angle neutron scattering studies by Castelletto et al. have recently provided strong evidence that the pH-responsive DPAMPC-DPA triblock copolymers form a three-dimensional

Figure 7. Light micrographs of V79 hamster lung cells cultured in 5 and 10% NIPAM81-MPC200-NIPAM81 copolymer gels showing increases in colony size after 24, 48, and 72 h. The scale bar is 80 µm.

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micellar gel network containing hydrophobic DPA-based domains.19 Given the translucent appearance of the thermoresponsive gels (see Figure 4), a similar micellar network is most likely formed by the NIPAM-MPC-NIPAM triblock copolymers. Small-angle X-ray and neutron scattering studies of the free-standing gels formed at higher copolymer concentrations are planned to determine the dimensions of the hydrophobic domains formed by the NIPAM chains. We have carried out preliminary rheological studies on these thermo-responsive NIPAM-MPC-NIPAM triblock copolymers. A typical viscosity versus temperature plot obtained at a shear stress of 1.00 Pa is shown in Figure 6 for an 8 wt % gel prepared using the NIPAM89-MPC250NIPAM89 triblock copolymer. At 20 °C this copolymer solution behaved as a Newtonian fluid, and below 30 °C the solution viscosity was less than 0.1 Pa‚s. A sharp increase in viscosity occurred at around 35 °C, indicating the onset of gelation. This observation agrees reasonably well with the light scattering data shown in Figure 4. At 37 °C the viscosity of the gel exceeded 100 Pa‚s, which is similar to that reported for related thermo-responsive PNIPAM-based gels.21 On cooling, some hysteresis is observed; the gel viscosity lags the solution viscosity by 2-3 °C (see Figure 6). The significant increase in viscosity that occurs at around 35 °C is consistent with the formation of a micellar network; the relatively low gel viscosity is not surprising given that these gels typically contain more than 92% water. Cell viability experiments confirmed that these thermoresponsive gels are sufficiently biocompatible to act as a culture medium for V79 cells (hamster lung cells).22 Figure 7 illustrates the increase in cell number with culture time within the 5 and 10% gels. The cells taken from these gels were shown to be viable for subsequent culture on tissue culture plastic. In contrast, cells did not survive when cultured in the 20% gel. We believe that this reduced cell viability is most likely due to the poor diffusion of nutrients and waste products from the cells through this more viscous gel. These preliminary studies suggest that the less viscous copolymer gels are worthy of further evaluation for the injectable delivery of cells within a biocompatible matrix for cell therapeutics, such as treatment of diabetes, and also tissue engineering applications such as cartilage regeneration. In summary, a series of reasonably well-defined novel ABA triblock copolymers were prepared via ATRP. The NIPAM-MPC-NIPAM triblock copolymers are new thermoresponsive biocompatible gelators that form physical gels reversibly over a fairly narrow temperature range under physiologically relevant conditions and at relatively low copolymer concentrations (