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Langmuir 2006, 22, 10180-10184
Rheological Studies of Thermosensitive Triblock Copolymer Hydrogels Tina Vermonden,*,† Nicolaas A. M. Besseling,‡ Mies J. van Steenbergen,† and Wim E. Hennink† Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht UniVersity, P.O. Box 80082, 3508 TB Utrecht, The Netherlands, and Laboratory of Physical Chemistry and Colloid Science, Wageningen UniVersity, Dreijenplein 6, 6703 HB Wageningen, The Netherlands ReceiVed July 28, 2006. In Final Form: September 6, 2006 Hydrogel formation by physical cross-linking is a developing area of research toward materials suitable for pharmaceutical and biomedical applications. Polymers exhibiting lower critical solution temperature (LCST) behavior in aqueous solution are used in this study to prepare hydrogels. Four triblock copolymers (ABA) with thermosensitive poly(N-(2-hydroxypropyl) methacrylamide lactate) A-blocks and a hydrophilic poly(ethylene glycol) B-block have been synthesized. The molecular weight of the hydrophilic PEG block was fixed at 10 kDa, whereas the molecular weight of the pHPMAm-lactate block was varied between 10 and 20 kDa. The rheological characteristics of these polymer hydrogels were studied as a function of temperature, concentration, and the length of the thermosensitive blocks. Gelation occurred rapidly upon increasing the temperature to 37 °C, which makes this system suitable as an injectable formulation. The gels became stronger with increasing temperature and concentration, and moreover they behaved as critical gels, which means that G′ and G′′ follow power laws over the entire frequency range. Surprisingly, with increasing length of the thermosensitive blocks, weaker hydrogels were formed. This trend can be explained by the cross-link density of the physical network, which increases with decreasing length of the thermosensitive blocks.
Introduction
* Corresponding author. Tel.: +31-30-2537304. Fax: +31-30-2517839. E-mail:
[email protected]. † Utrecht University. ‡ Wageningen University.
Temperature sensitive polymers are very suitable candidates for the A-blocks in ABA triblock copolymers for the development of hydrogels for pharmaceutical and biomedical applications.10-13 Polymers that display LCST behavior (lower critical solution temperature) dissolve in water below the cloud point (CP) and become insoluble above this temperature due to dehydration of the polymer chains. Poly(N-(2-hydroxypropyl)methacrylamide lactate (poly(HPMAmlac)) is an interesting thermosensitive polymer because the cloud point can be tuned by the number of lactate groups attached to the HPMAm monomers.14 In addition, the lactate groups are hydrolyzed in physiological conditions, which in turn is associated with an increase of the cloud point.15 Once enough lactate groups have been hydrolyzed, the polymer becomes soluble at 37 °C. PolyHPMAm is a nontoxic polymer with a good biocompatibility, and several copolymers of polyHPMAm are currently being evaluated clinically.16 Poly(HPMAmlac) has already been used as A-block in ABblock copolymers with poly(ethylene glycol) as B-blocks, which are able to form polymeric micelles.15 Above the cloud point and at a sufficient polymer concentration, aqueous solutions of triblock copolymers with poly(HPMAmlac) as A-blocks and a biocompatible hydrophilic polymer, such as poly(ethylene glycol) (PEG) as the B-block, yield a network. The mechanism of self-assembly of these hydrogels is shown in Figure 1. In situ gel formation makes these polymer solutions promising injectable biomaterials
(1) Lee, Y. K.; Mooney, D. J. Chem. ReV. 2001, 101, 1869-1879. (2) Hoffman, A. S. AdV. Drug DeliVery ReV. 2002, 43, 3-12. (3) Hennink, W. E.; Van Nostrum, C. F. AdV. Drug DeliVery ReV. 2002, 54, 13-36. (4) Tew, G. N.; Sanabria-DeLong, N.; Agrawal, S. K.; Bhatia, S. R. Soft Matter 2005, 1, 253-258. (5) Molina, I.; Li, S.; Bueno Martinez, M.; Vert, M. Biomaterials 2001, 22, 363-369. (6) Li, S. Macromol. Biosci. 2003, 3, 657-661. (7) Yu, J. M.; Dubois, P.; Teyssie´, P.; Je´roˆme, R.; Blacher, S.; Brouers, F.; L’Homme, G. Macromolecules 1996, 29, 5384-5391. (8) Aamer, K. A.; Sardinha, H.; Bhatia, S. R.; Tew, G. N. Biomaterials 2004, 25, 1087-1093. (9) Vermonden, T.; Van Steenbergen, M. J.; Besseling, N. A. M.; Marcelis, A. T. M.; Hennink, W. E.; Sudho¨lter, E. J. R.; Cohen Stuart, M. A. J. Am. Chem. Soc. 2004, 126, 15802-15808.
(10) Peppas, N. A.; Bures, P.; Leobandung, W.; Ichikawa, H. Eur. J. Pharmacol. Biopharm. 2000, 50, 27-46. (11) Ruel-Garie´py, E.; Leroux, J.-C. Eur. J. Pharmacol. Biopharm. 2004, 58, 409-426. (12) Jeong, B.; Kim, S. W.; Bae, Y. H. AdV. Drug DeliVery ReV. 2002, 54, 37-51. (13) Lee, J.; Bae, Y. H.; Sohn, Y. S.; Jeong, B. Biomacromolecules 2006, 7, 1729-1734. (14) Soga, O.; Van Nostrum, C. F.; Hennink, W. E. Biomacromolecules 2004, 5, 818-821. (15) Soga, O.; Van Nostrum, C. F.; Ramzi, A.; Visser, T.; Soulimani, F.; Frederik, P. M.; Bomans, P. H. H.; Hennink, W. E. Langmuir 2004, 20, 93889395. (16) Duncan, R. Nat. ReV. Drug DiscoVery 2003, 2, 347-360.
Amphiphilic polymers have been studied extensively in the past decade with the purpose of constructing hydrogels as biomaterials for drug delivery or tissue engineering applications.1-3 In particular, triblock copolymers with an ABA-architecture of hydrophobic A-blocks and hydrophilic B-blocks are suitable molecules to construct hydrogels.4-6 In aqueous environment, association of the hydrophobic blocks yields the formation of micelles above the critical micelle concentration (cmc), whereas at much higher concentrations bridging between micelles occurs, which results in the formation of a dynamic network. Because the B-blocks are hydrophilic, the hydrogels can retain large water contents, and in that respect they mimic natural tissues. They are highly permeable to mobile ions and other small molecules, which makes rapid transport of nutrients and cellular waste compounds and polymer degradation products possible. The mechanical properties of the hydrogels can be tuned by changing different parameters such as the kind of monomers that are used, the length of the different blocks, and external parameters such as the polymer concentration, pH, and temperature.7-9
10.1021/la062224m CCC: $33.50 © 2006 American Chemical Society Published on Web 10/25/2006
ThermosensitiVe Triblock Copolymer Hydrogels
Langmuir, Vol. 22, No. 24, 2006 10181 Table 1. Characteristics of pHPMAmlac-PEG-pHPMAmlac Block Copolymers: 10, 16, 18, and 20 10 16 18 20
Figure 1. Schematic representation of hydrogel formation as a function of temperature.
a
Mna (kDa)
Mnb (kDa)
Mwb (kDa)
Mw/Mnb
10-10-10 16-10-16 18-10-18 20-10-20
19 23 30 43
31 40 62 67
1.63 1.71 2.11 1.57
Determined by 1H NMR. b Determined by GPC.
Figure 2. Structure of pHPMAmlac-PEG-pHPMAmlac block copolymers 10, 16, 18, and 20.
when the cloud point of a polymer solution is between room temperature and body temperature. The rheological characteristics of hydrogels are very important for their applications. Rheological behavior of aqueous solutions of triblock copolymers with PEG as the hydrophilic middle block have been studied mainly for polymers with small hydrophobic end groups, such as linear hydrocarbon chains.17,18 Only a few rheological studies using biodegradable polylactides8 and thermosensitive poly(N-isopropylacrylamides)19 as hydrophobic blocks have been reported. In this study, we report the rheological properties of hydrogels prepared from ABA triblock copolymers containing PEG B-blocks with a molecular weight of 10 kDa and pHPMAmlac A-blocks with molecular weights varying between 10 and 20 kDa. For hydrogels prepared at different concentrations, the storage and loss modulus (G′ and G′′) were measured for a range of frequencies and temperatures. We use the gel model developed by Winter and co-workers to analyze the hydrogel properties.20,21 As discussed below, the strength of a hydrogel is influenced by both the polymer concentration and the molecular weight of the A-blocks in the polymers.
Results and Discussion Block Copolymer Synthesis. pHPMAmlac-PEGpHPMAmlac block copolymers were synthesized by radical polymerization using HPMAm-monolactate and HPMAmdilactate as monomers and PEG-ABCPA as macroinitiator with yields between 65% and 75%. The structure and the molecular characteristics of the polymers are shown in Figure 2 and Table 1. Four block copolymers with different pHPMAmlac block lengths were synthesized by changing the ratio of monomer to macroinitiator, while the macroinitiator concentration was kept constant. The molecular weight of the PEG block was fixed at 10 kDa, whereas the molecular weights of the hydrophobic blocks in the polymers were 10, 16, 18, and 20 kDa as determined by 1H NMR measurements (further referred to as polymers 10, 16, 18, and (17) Pham, Q. T.; Russel, W. B.; Thibeault, J. C.; Lau, W. Macromolecules 1999, 32, 5139-5146. (18) Walderhaug, H.; Hansen, F. K.; Abrahmse´n, S.; Persson, K.; Stilbs, P. J. Phys. Chem. 1993, 97, 8336-8342. (19) Aubry, T.; Bossard, F.; Staikos, G.; Bokias, G. J. Rheol. 2003, 47, 577587. (20) Scanlan, J. C.; Winter, H. H. Macromolecules 1991, 24, 47-57. (21) Izuka, A.; Winter, H. H.; Hashimoto, T. Macromolecules 1992, 25, 24222428.
Figure 3. Storage modulus G′ (black diamonds) and loss modulus G′′ (gray squares) as a function of time for polymer 16 (26% (w/w)) in 120 mM ammonium acetate buffer pH 5.0 at a frequency of 1 Hz and 1% strain during quick heating from 5 °C (at 0 min) to 37 °C (at 1.1 min).
20, respectively). The molecular weights calculated using 1H NMR measurements show the same trends as the molecular weights measured using GPC (see Table 1). For GPC measurements, PEG-standards were used to determine the molecular weights. Using these standards, the number average molecular weights (Mn) of the triblock copolymers are underestimated when compared to the results of the NMR measurements. Generally, the molecular weights of triblock copolymers, as determined by GPC, might result in an under- or overestimation of the molecular weight, because calibration is performed with homopolymers. The feed ratio of HPMAm-monolactate/HPMAm-dilactate was kept constant at 25/75. NMR analysis showed that in all polymers the ratio ML/DL was about 50/50, which is higher than the feed ratio. Apparently, the smaller HPMAm-monolactate reacts faster than the larger HPMAm-dilactate monomer. All of the polymers displayed LCST behavior with a cloud point between 15 and 20 °C. At this temperature, the scattering intensity of samples of about 3 mg/mL increased rapidly. Upon cooling, a clear solution was obtained again, proving the reversible process of association. Rheological Characterization of Hydrogels. The kinetics of gel formation was studied in a time sweep experiment while the temperature was increased rapidly (Figure 3). This figure shows G′ and G′′ as a function of time for polymer 16 at a concentration of 26% (w/w) in an ammonium acetate buffer of pH 5.0. This pH was chosen to minimize hydrolysis of the lactate side chains of the polymers during the experiments.22 At the start of the experiment (time ) 0 min), the temperature was 5 °C, and in approximately 1 min the temperature was increased to 37 °C. Both G′ and G′′ increase rapidly with increasing temperature, and G′ was already larger than G′′ when the temperature reached 37 °C (time ∼1 min). Tan δ ()G′′/G′) decreased from 1.7 at 5 °C to 0.6 at 37 °C, indicating a transition from a viscoelastic fluid to a solid gel. As soon as the temperature was stable at 37 °C, also G′ and G′′ remained constant, indicating that a stable (22) Neradovic, D.; Van Steenbergen, M. J.; Vansteelant, L.; Meijer, Y. J.; Van Nostrum, C. F.; Hennink, W. E. Macromolecules 2003, 36, 7491-7498.
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Vermonden et al.
than G′′ over the whole frequency range and importantly tan δ ()G′′/G′) is independent of the frequency. This behavior is typical for a critical gel, which is an intermediate state between liquid and solid.23 A critical gel is characterized by power law behavior in which G′ and G′′ can be described with eq 1. Tan δ ()tan(nπ/2)) is independent of frequency but proportional to the relaxation exponent n.20,21
G′(ω) )
Figure 4. Storage modulus G′ (black) and loss modulus G′′ (gray) as a function of temperature for polymer 20 (29% (w/w)) in 120 mM ammonium acetate buffer pH 5.0 at a frequency of 1 Hz and 1% strain.
Figure 5. Storage modulus G′ (black diamonds), loss modulus G′′ (gray squares), and tan δ (open circles) as a function of the frequency ω for a hydrogel of polymer 16 of 32% (w/w) in 120 mM ammonium acetate buffer pH 5.0 at 1% strain at 40 °C.
gel was formed. Similar curves were obtained for polymers 18 and 20 with a concentration of at least 25% (w/w). Even at a high concentration (35% (w/w)), it was not possible to prepare hydrogels with polymer 10. Apparently, the molecular weight of the hydrophobic blocks is too small for the polymers to selfassemble into a network. The rate of gel formation upon increasing temperature was also measured for a few samples at a physiological pH (pH ) 7.2, 0.2 M HEPES buffer). Because changing the pH does not generate charged polymers, which could interfere with the gelation mechanism, no significant differences in gel formation are expected. Indeed, the gel formation was not influenced by increasing the pH to 7.2. Because hydrolysis of the lactate side chains occurs in time at pH 7.2 and the rheology experiments take several hours per sample, further rheology experiments were performed at pH 5.0 to minimize the hydrolysis of the lactic acid side chains.15,22 Figure 4 shows the temperature dependence of G′ and G′′ of triblock copolymer 20 in buffer. The storage modulus G′ increases with increasing temperature, and at 19 °C, G′ equals G′′ (gel point). This temperature is only 1-2 °C higher than the cloud point measured using scattering measurements at equal heating rates. The small difference is probably caused by the much lower concentrations used in the scattering experiments (0.3% versus 29% (w/w)). Above the gel point, G′ slightly increases and reached at plateau value at approximately 40 °C. The hydrogels prepared from polymers 16 and 18 yielded curves with a similar shape. To further characterize the hydrogels and compare the properties of the polymers with different hydrophobic block lengths, the storage and loss modulus were measured as a function of the frequency at various temperatures. Figure 5 shows that above the cloud point of the thermosensitive block G′ is larger
G′′(ω) ) Γ(1 - n) cos(nπ/2)Sωn tan(nπ/2)
(1)
S is called the strength of the gel, Γ() is the gamma function, ω is the frequency, and n is the relaxation exponent with a value between 0 and 1. The value for n can be found as the slope of the parallel straight lines for the log-log frequency dependence of G′ and G′′ (G′ ≈ G′′ ≈ ωn), whereas the distance between the lines is given by G′′/G′ ) tan(nπ/2). The frequency dependence of G′ and G′′ of the hydrogels could be fitted to eq 1 at any temperature above the cloud point. Hence, the hydrogels behaved as critical gels at all of these conditions, although power law behavior is usually found only at one condition (the gel point). Only a few other examples of gels exhibiting power law behavior at more than one condition have been reported.8,24 For the exponent n, different values have been reported for both chemically cross-linked and physically cross-linked networks. For chemically cross-linked gels, these values usually correspond to theoretically predicted values of n between 0.67 and 1 (using Rouse dynamics or mean field theory).7,20 For physically cross-linked gels, a larger range of values for the scaling exponent n has been found experimentally (between 0.1 and 0.8).8,25 In our study, we found values for n between 0.25 and 0.65 depending on the temperature. With increasing temperature, smaller values for n were found for the hydrogels. Earlier, the relationship between the scaling exponent n and the extent of cross-linking was described for a chemically cross-linked polyethylene gel, which displays power law behavior in a broad vicinity of the gel point.26 With increasing extent of cross-linking, smaller values for n were found. Possibly, a higher extent of (physical) cross-linking is also present in our hydrogels at higher temperatures, resulting in lower values for n. At higher extent of cross-linking, also stronger gels can be expected. Therefore, it is not surprising that with increasing temperature not only the exponent n decreases, but also the gel strength S increases. The relationship between S and n is given in eq 2, where G0 is the material modulus and t0 is a relaxation time constant.20,21
S ) G0‚t0n
(2)
Hence, from a plot of log S versus n (Figure 6), the material modulus G0 and the relaxation time t0 for each polymer hydrogel could be determined using eq 2 (Table 2). Hydrogels were prepared at different concentrations, and these results are displayed in Figure 6A-C. Not surprisingly, with increasing concentrations, increasing gel strengths S were found. Figure 6 also shows that the gel strength S for hydrogels of the same concentration increases when the molecular weight of the hydrophobic blocks of the polymers decreases. This is in (23) Larson, R. G. The Structure and Rheology of Complex Fluids; Oxford University Press: New York, 1999. (24) Sivakova, S.; Bohnsack, D. A.; Mackay, M. E.; Suwanmala, P.; Rowan, S. J. J. Am. Chem. Soc. 2005, 127, 18202-18211. (25) Matsumoto, T.; Kawai, M.; Masuda, T. Macromolecules 1992, 25, 54305433. (26) Valle´s, E. M.; Carella, J. M.; Winter, H. H.; Baumgaertel, M. Rheol. Acta 1990, 29, 535-542.
ThermosensitiVe Triblock Copolymer Hydrogels
Langmuir, Vol. 22, No. 24, 2006 10183
Figure 7. Schematic representation of the influence of the length of the thermosensitive blocks on the cross-link density of the network in the hydrogels.
Figure 8. Material modulus (G0) as a function of concentration for 16 (black circles), 18 (gray triangles), and 20 (black squares) hydrogels in 120 mM ammonium acetate buffer pH 5.0.
Figure 6. Gel strength S as a function of the relaxation exponent n for hydrogels of triblock copolymers 16 (black circles), 18 (gray triangles), and 20 (black squares) at different concentrations. Table 2. Material Modulus and Relaxation Time of Hydrogels at Different Concentrationsa hydrogel 26% 29% 32%
16 18 20 16 18 20 16 18 20
G0 (kPa)
t0 (ms)
2.5 2.1 1.1 4.9 3.3 2.8 12.9 9.5 8.5
2.4 2.0 1.5 2.3 3.1 2.1 2.3 2.3 2.1
a Typical errors found: G ( 1.0 kPa, and t ( 1.0 ms. Typical 0 0 correlation coefficients (R) of the fits are 0.98.
contrast to expectations and can be explained as follows. For diblock copolymers of PEG-pHPMAlac, which associate into micelles, it was found that the aggregation number decreases when the hydrophobic block length decreases.15 The relationship between the aggregation number and the hydrophobic block length in micelles for diblock copolymers is expected to correlate with the aggregation number in the physical cross-links of the network for our triblock copolymer hydrogel. So, if indeed a smaller number of molecules can associate in one cross-link for the shorter polymers, the total number of cross-links per volume must be larger than for the longer polymers at equal concentrations as shown schematically in Figure 7. A similar correlation between the parameter S and the cross-link density was reported earlier for polyurethane gels at the gel point.27 In Table 2, the fitted rheological parameters for the hydrogels at the different concentrations are summarized. The data were
fitted using eq 2 with correlation coefficients (R) close to 1, which confirm the good fits. The material modulus G0 shows the same trend as the parameter S. G0 increases with increasing concentration and decreases with increasing molecular weight of the polymers. The relaxation time t0 is more or less constant at 2 ms for all polymers at any concentration, which is also reflected in the constant slopes in the S-n plots in Figure 6. Figure 8 shows the material modulus G0 as a function of concentration for the three triblock polymers investigated. The data can be fitted with lines with slopes of 5.1 ( 1.0, 4.7 ( 1.5, and 6.5 ( 0.8 for hydrogels containing polymers 16, 18, and 20, respectively. Scaling exponents of G0 as a function of concentration between 5.0 and 5.6 have been reported for networks of nanodroplets in w/o microemulsions connected by triblock copolymers of PEG-polyisoprene-PEG structure.28 These triblock copolymers have hydrophilic outer blocks and a hydrophobic inner block, which gives them the reversed structure when compared to our polymers. Because the gel formation of these polymers occurred in oil media instead of water, the gel formation processes are similar. In both cases, the outer blocks self-assemble and form the cross-links in the network, whereas the inner blocks are solvated. Scaling exponents between 5 and 5.6 were found only in a concentrated regime. The scaling exponents of G0 of our hydrogels (between 4.7 and 6.5), also obtained at high concentrations, correspond nicely with the values found previously for the reversed triblock copolymer system.
Concluding Remarks In this paper, we presented the rheological properties of thermoreversible hydrogels prepared from amphiphilic triblock copolymers. The hydrogels behaved as critical gels above the (27) Chambon, F.; Petrovic, Z. S.; MacKnight, W. J.; Winter, H. H. Macromolecules 1986, 19, 2146-2149. (28) Zo¨lzer, U.; Eicke, H.-F. J. Phys. II, France 1992, 2, 2207-2219.
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cloud point. With increasing concentration, stronger gels were formed. Furthermore, the strengths of the gels were negatively correlated to the molecular weight of the temperature sensitive end blocks. This is caused by the higher cross-link density for networks of shorter polymers. For pharmaceutical applications, it is important to know that when the temperature of the injected polymer solution is below the cloud point, gel formation occurs very fast in vivo. This is necessary to prevent dilution of the polymer solution, as this would cause too fast release of any bioactive components enclosed in the gel. The gels are formed immediately upon increasing the temperature to 37 °C, which makes it possible to use them as injectable formulations. At the moment, the degradation of the hydrogels in vitro and biocompatibility in vivo are being studied. Experimental Section All commercial chemicals were obtained from Aldrich and were used as received. L-Lactide was obtained from Purac Biochem BV (Gorinchem, The Netherlands). HPMAm-monolactate and HPMAmdilactate were synthesized according to the method described previously.22 The Mn of PEG (Mw ) 10 000 g/mol according to the supplier) was determined by reaction of the end groups with trichloroacetyl isocyanate (TAIC) according to a reported method (Mn ) 9970).29 A PEG-4,4′azobis(4-cyanopentanoic acid) macroinitiator (PEG-ABCPA) was synthesized using a procedure similar to that of Neradovic et al.30 The molecular weights of the obtained triblock copolymers were determined by GPC using a Plgel 5 µm MIXED-D column (Polymer Laboratories). Poly(ethylene glycol)s of defined molecular weight were used as standards. The eluent was DMF containing 10 mM LiCl, the elution rate was 0.7 mL/min, and the column temperature was 40 °C. The concentration of the samples was 5 mg/mL in 10 mM LiCl in DMF. 1H NMR (300 MHz) spectra were recorded on a Varian Mercury Plus 300 spectrometer using CDCl3 as a solvent. All experiments were performed in a 120 mM ammonium acetate buffer pH 5.0. For the experiments at a higher pH, a 0.2 M 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid buffer of pH 7.2 was (29) Loccufier, J.; Van Bos, M.; Schacht, E. Polym. Bull. (Berlin) 1991, 27, 201-204. (30) Neradovic, D.; Van Nostrum, C. F.; Hennink, W. E. Macromolecules 2001, 34, 7589-7591.
Vermonden et al. used. The cloud point of the polymers was measured with light scattering using a Horiba Fluorolog fluorometer (650 nm, 90° angle). The polymers were dissolved at a concentration of 3 mg/mL, and the scattering intensity was measured every 0.2 °C during heating with a rate of approximately 1 °C/min. The rheology experiments were performed on a TA Instruments AR1000-N rheometer using a cone-plate geometry (steel, 20 mm diameter with an angle of 1°). A solvent trap was used to prevent evaporation of the solvent. The temperature-dependent experiments were performed with a temperature ramp of 1 °C/min. The concentrations are given in % w/w. Synthesis of Triblock Copolymer, 20. 0.64 g of PEG-ABCPA macroinitiator, 0.60 g (2.79 mmol) of HPMAm-lac, and 2.41 g (8.40 mmol) of HPMAm-lac2 (molar ratio mono-/dilactate 25/75) were dissolved in 10 mL of dry acetonitrile in an airtight screw-cap glass vial. This reaction mixture was flushed with nitrogen for 15 min. Subsequently, the solution was stirred for 40 h at 70 °C. The polymer was purified by dialysis against water at 4 °C for 4 days (MWCO 12-14 kDa) and obtained as a white solid after freeze-drying. Yield: 2.6 g, 71%. 1H NMR (CDCl3): δ 6.5 (1H, b, NH), 5.1 (2H, b, NHCH2CH(CH3)O and COCH(CH3)O), 4.4 (1H, b, COCH(CH3)OH), 3.7 (904H, s, OCH2CH2 (PEG-protons)), 3.1 (2H, b, NHCH2), 2.2-0.6 (main chain protons and nCH3 of HPMAm-lacn). The copolymer composition was determined by 1H NMR from the ratio of the integral of the peaks at 5.1 (I5.1) and 4.4 (I4.4) ppm using eq 3. %HPMAm-lac2 )
(
)
I5.1 - 1 × 100% I4.4
(3)
The number average molecular weight can be determined from the NMR spectrum using eq 4 and the average molecular weight of the monomers (Mw,ave,HPMAm-lacn). Mn )
Mw,ave,HPMAm-lacn‚I4.4 I3.7/904
(4)
The triblock copolymers with smaller thermosensitive blocks were synthesized using the same procedure; only the ratio of HPMAlacn/PEG-ABCPA was smaller. The ratio of HPMA-lac/HPMAlac2 was kept constant.
Acknowledgment. This research was supported by a grant from the Dutch Program for Tissue Engineering (Project Number 6731). LA062224M
