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pH and Temperature-Sensitive N-Isopropylacrylamide Ampholytic Networks Incorporating L-Lysine Marcin Karbarz, Karolina Pulka, Aleksandra Misicka, and Zbigniew Stojek* Department of Chemistry, Warsaw UniVersity, Pasteura 1, PL-02-093 Warsaw, Poland ReceiVed February 3, 2006. In Final Form: June 10, 2006 Ampholytic polymer gels based on N-isopropylacrylamide (NIPA) and natural amino acid L-lysine were prepared by free radical polymerization in aqueous solutions. To make amino acids attachable to the polymer chain, the acrylic group was added to the -amino group of lysine to obtain N--acrylic-lysine (Z). Finally, a new temperature- and pH-sensitive (NIPA-Z) hydrogel was obtained. The presence of amino and carboxylic groups of amino acids gave us a possibility to control the amount and sign of the excessive charge on the polymeric network with respect to pH. The swelling behavior of the NIPA-Z hydrogels with respect to the amount of Z (0-4%), temperature, and pH was investigated. Temperature and pH were changed in the ranges of 20-50 °C and 2-12, respectively. To eliminate the influence of ionic strength on the swelling behavior, this parameter was kept constant in all experiments. It was found that the pH dependence of the degree of swelling for the NIPA-Z gels, measured at constant temperature, exhibits a minimum. Such a minimum was seen for the ampholyte networks with independent acidic and basic groups attached to the polymer chains. For the NIPA-Z gels, the minimum was wide, and the pH range over which it was spread corresponded well to the pH distribution of the zwitterions. The way the gel volume changed with an increase in temperature depends on the amino acid amount. It is initially discontinuous and turns to the continuous one for the higher percentage of amino acid. The temperature dependence of the swelling process obtained for different pH values clearly shows that for the pH region where the zwitterions dominate, the polymer networks collapse more efficiently.
Introduction Many polymer gels undergo reversible volume changes. These changes take place in response to a shift in the balance between repulsive intermolecular forces (they make the polymer network expand) and attractive forces that make it shrink. Repulsive forces are usually electrostatic interactions between the groups that have the same charge. The osmotic pressure also plays an important role in expanding the polymer networks. Attractive interactions can be those of the van der Waals type, hydrophobic interactions, hydrogen bonding, and electrostatic interactions between the groups of opposite charge. The volume phase transition occurs as a response to the changes in, for example, temperature,1-3 pH,3-6 ionic strength,6 pressure,7 solvent composition,8 and electromagnetic radiation.9 The theoretical studies and predictions on that phenomenon date back to 1968.10 Then, Dusek and Patterson predicted that when external forces were applied to the gel, the volume of those gels might undergo discontinuous change. Experimental evidence of such a discontinuous phase transition was provided several years later.11 Because of their environmental sensitivity and unique structure, these polymeric hydrogels have been investigated for many biomedical and pharmaceutical applications including drug delivery systems, separation techniques, muscle-like actuators, and construction of sensors.12-19 Among these intelligent * Corresponding author. Tel.: (48-22) 822 0211 ext. 336; fax: (48-22) 822 4889; e-mail:
[email protected]. (1) Zhang, X. Z.; Zhang, J. T.; Zhuo, R. X.; Chu, C. C. Polymer 2002, 43, 4823. (2) Karbarz, M.; Gniadek, M.; Stojek, Z. Electroanalysis 2005, 17, 1396. (3) Hoare, T.; Pelton, R. Macromolecules 2004, 37, 2544. (4) Kang, S. I.; Bae, Y. H. Macromolecules 2001, 34, 8173. (5) Annaka, M.; Tanaka, T. Nature 1992, 355, 30. (6) Ogawa, K.; Nakayama, A.; Kokufuta, E. Langmuir 2003, 19, 3178. (7) Kato, E. J. Chem. Phys. 1997, 106, 3792. (8) Harland, R. S.; Prudhomme, R. K. Polyelectrolyte Gels. Properties, Preparation, Application; American Chemical Society: Washington, DC, 1992. (9) Suzuki, A.; Tanaka, T. Nature 1990, 346, 345. (10) Dusek, K.; Patterson, D. J. Polym. Sci., Polym. Phys. Ed. 1968, 6, 1209. (11) Tanaka, T.; Fillmore, D. J.; Nishio, I.; Swislow, G.; Shah, A. Phys. ReV. Lett. 1980, 45, 1636.
polymers, pH- and temperature-sensitive hydrogels are the most widely investigated. It is commonly known that a group of polymeric hydrogels (i.e., poly-(N-isopropylacrylamide) (NIPA) gels) exhibits a drastic swelling transition at its lower critical solution temperature (LCST) of 34 °C. At temperatures below 34 °C, the gels are swollen, whereas at temperatures higher than 34 °C, the gels dehydrate to the collapsed state. By introducing the groups to the polymeric chains, which are able to change their charge in response to a change in pH (weak acid, base, or their salts), the NIPA gel acquires new properties such as sensitivity to a change in pH, enhancement of the volume change during the volume phase transition, increase in the temperature at which the phenomenon starts, and a possibility of switching the volume phase transition from a discontinuous to a continuous one.20-22 Previous studies on temperature- and pH-sensitive gels usually involved polymers prepared from monomers that were either acidic or basic.23 In this work, we describe the swelling behavior of the gels that are modified with a species that has both acidic and basic groups (zwitterions). Zwitterions were also built into linear polymeric systems.24 The incorporation of zwitterions has one advantage as compared to separate acidic and basic molecules: the same total number of ionized groups excludes less neutral monomers in the chain when the zwitterions are (12) Zhang, J. T.; Huang, S. W.; Liu, J.; Zhuo, R. X. Macromol. Biosci. 2005, 5, 192. (13) Mahkam, M. J. Biomed. Mater. Res. 2005, 75B, 108. (14) Hyk, W.; Karbarz, M.; Stojek, Z.; Ciszkowska, M. J. Phys. Chem. B 2004, 108, 864. (15) Konishi, Y.; Asai, S.; Midoh, Y.; Oku, M. Sep. Sci. Technol. 1993, 28, 1691. (16) Kim, H. I.; Park, S. J.; Kim, S. I.; Kim, N. G.; Kim, S. J. Synth. Metals 2005, 155, 674. (17) Hinkley, J. A.; Morgret, L. D.; Gehrke, S. H. Polymer 2004, 45, 8837. (18) Ogawa, K.; Wang, B.; Kokufut, E. Langmuir 2001, 17, 4704. (19) Holtz, J. H.; Asher, S. A. Nature 1997, 389, 829. (20) Kawasaki, H.; Sasaki, S.; Maeda, H. J. Phys. Chem. B 1997, 101, 5089. (21) Kawasaki, H.; Sasaki, S.; Maeda, H. J. Phys. Chem. B 1997, 101, 4184. (22) Motonaga, T.; Shibayama, M. Polymer 2001, 42, 8925. (23) Kudaibergenov, S. E. AdV. Polym. Sci. 1999, 144, 115. (24) Lowe, A. B.; McCormick, C. L. Chem. ReV. 2002, 102, 4177.
10.1021/la060334n CCC: $33.50 © 2006 American Chemical Society Published on Web 08/01/2006
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Figure 1. Scheme of the synthesis of N--acrylic lysine.
involved. We have employed L-lysine, a natural amino acid. To make it attachable to the polymer chain, we added the acrylic group to the -amino group of lysine. Eventually, a new temperature- and pH-sensitive (NIPA-Z) hydrogel was synthesized. The presence of amino and carboxylic groups of amino acids gave us a possibility to control the charge on the polymeric network in the function of pH. At a low pH, the positive form (-NH3+) dominates. For a pH close to the isoelectric point (pI), mostly an internal salt called a zwitterion is present (-NH3+ and -COO-). Note that the zwitterion has no net charge. For high pH values, the negative form (-COO-) dominates. We have focused our investigation on the dependence of the swelling behavior of the polymer in aqueous solutions on temperature and pH. To avoid the influence of ionic strength on the swelling behavior, this parameter was set at the same level in all experiments. Uncharged NIPA gels served as the reference point. Experimental Procedures Materials. Polymer constituents: N-isopropylacrylamide (NIPA, 97%), N,N′-methylenebisacrylamide (BIS, 99%), ammonium persulfate (APS, 99.99%), and N,N,N′,N′-tetramethylethylenediamine (TEMED, 99.5%) were purchased from Aldrich. Reagents for the synthesis of the -acrylic derivative of lysine: 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 98%), N,N′-diisopropylcarbodiimide (DIC, 99%), 4-(dimethylamino)pyridine (DMAP, 99%), and N,Ndiisopropylethylamine (DIPEA, 99%) were also purchased from Aldrich. N-R-t-butyloxycarbonyl-N--fluorenylmethyloxycarbonylL-lysine (Boc-Lys(Fmoc)-OH) was purchased from Bachem, and trifluoroacetic acid (TFA, 99%) was from Merck. All chemicals were used as received except for NIPA, which was recrystallized twice from the benzene/hexane mixture (90:10 v/v). All solutions were prepared using high purity water obtained from a Milli-Q Plus/Millipore purification system (conductivity of water: 0.056 µS cm-1). Synthesis of N--Acrylic Derivative of Lysine (Z). The desired compound was obtained from Boc-Lys(Fmoc)-OH in four steps (see Figure 1). (a) Synthesis of Methyl Ester of Boc-Lys(Fmoc)-OH.25 BocLys(Fmoc)-OH (2.21 g, 4.7 mmol) was suspended in DCM (20 mL). Then, DMAP (0.29 g, 2.35 mmol) and MeOH (220 mL, 5.34 mmol) were added. The reaction mixture was stirred and kept at 0 °C (ice bath), and after 10 min, DIC (739 mL, 4.7 mmol) was added. The ice bath was removed, and the reaction mixture was stirred overnight at room temperature. The precipitate (N,N′-diisopropylurea) was filtered out, and the filtrate was washed with saturated NaHCO3 (15 mL × 3), 10% citric acid (15 mL × 3), and saturated NaCl (15 mL × 3). The organic layer was dried over MgSO4 and evaporated to leave a white solid. The product was taken to the next step without purification; mass spectrometry confirmed the molar mass of the product. (b) Synthesis of Boc-Lys(NH2)-Ome.26 To a solution of BocLys(Fmoc)-OMe (1 g, 2.07 mmol) in THF (20 mL), 1-hexanethiol (25) Dhaon, M. K.; Olsen, R. K.; Ramasamy, K. J. Org. Chem. 1982, 47, 1962. (26) Sheppeck, J. E., II; Kar, H.; Hong, H. Tetrahedron Lett. 2000, 41, 5329.
Figure 2. Schematic structure of N-isopropylacrylamide-co--acrylic lysine polymer cross-linked with N,N′-methylenebisacrylamide. (1.5 mL, 10.35 mmol) was added, which was followed by dropwise addition of catalytic DBU (31 µL, 0.207 mmol). After 4.5 h, the solvent from the reaction mixture was evaporated. A dark-orange oil was obtained. It was purified by flash chromatography (CHCl3MeOH) to yield a yellow oil (0.47 g). (c) Synthesis of Acrylic Derivative of Boc-Lys(NH2)-OMe. To a solution of Boc-Lys(NH2)-OMe (0.47 g, 1.81 mmol) in AcOEt (15 mL), DIPEA (317 µL, 1.81 mmol) and acrylic acid (187 µL, 2.7 mmol) were added, and this was followed by the addition of DIC (284 µL, 1.81 mmol). After 24 h, the precipitate was filtered, and the filtrate was washed with saturated NaHCO3 (10 mL × 3), 10% citric acid (10 mL × 3), and saturated NaCl (10 mL × 3). The organic layer was dried over MgSO4. The product was taken to the next step without purification; mass spectrometry confirmed the molar mass of the product. (d) Synthesis of N--Acrylic-lysine (Z). Boc-Lyc(acrylic)-OMe (0.54 g, 1.72 mmol) was dissolved in MeOH (15 mL), and 4 M NaOH (650 mL) was added. After 24 h, the mixture was diluted with H2O, and MeOH was evaporated. The pH was adjusted to 3 with 10% citric acid. The aqueous layer was extracted with AcOEt (15 mL × 3). The combined organic layers were dried over MgSO4. After AcOEt evaporation, the oil was dissolved in a mixture of TFA/DCM (1:1, 5 mL). The stirring was continued for 2 h. Then, Et2O was added to precipitate the crude product. The crude product was purified by crystallization from MeOH/Et2O. The obtained NMR and MS spectra confirmed the structure of N--acrylic-lysine. MS [M + H]+ ) 201.2. 1H NMR (200 MHz, D2O): δ 6.2 (m; 2H; vinyl), 5.74 (dd; J ) 10 Hz, J ) 2.4 Hz; 1H; vinyl), 3.74 (t; J ) 6 Hz; 2H; -H), 3.28 (m; 1H; R-H), 1.85 (m; 2H; δ-H), 1.58 (k; J ) 6.6 Hz; 2H; β-H), 1.41 (m, 2H, γ-H). Synthesis of Poly(N-isopropylacrylamied)/N--acrylic Lysine (NIPA-Z) Hydrogels. NIPA-Z gels were synthesized by free-radical solution copolymerization. The total concentration of NIPA, Z, and BIS was kept constant at the 700 mM level. The concentration of BIS in all samples was fixed to be 7 mM, while the concentrations of NIPA and Z were varied. The pre-gel solutions were degassed, and polymerization was initiated and accelerated by APS (1.88 mM) and TEMED (32 mM) and carried out at 5 °C for 20 h. The gels with a mole fraction of the derivative of lysine equal to y, where y)
nz 100% nNIPA + nz + nBIS
in a pre-gel solution are denoted as y NIPA-Z. In this paper, y ) 4, 2, 1, 0.5, and 0% and refers only to the synthesis mixture. The gels were synthesized in glass capillaries of inner diameter, i.d., 340 µm. The samples of gels were cut into pieces of ca. 3 mm in length and immersed into pure water to remove unreacted residues. The schematic structure of the cross-linked NIPA-Z polymer is shown in Figure 2. Measurements of Dimensions of Gel Rods. After removing the unreacted residues, the gel samples were inserted into a water-jacketed
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Figure 3. pH dependence of degree of swelling for NIPA-Z gels measured at 25 °C. y ) 2% (gray circles) and 4% (black squares). cell filled with aqueous solutions of different pHs. The pH was changed by adding HCl or KOH and was controlled with a pH/ion meter (MeterLab, model PHM 240). To avoid changes in pH due to carbon dioxide content in the air, all experiments were done under argon atmosphere. In all experiments, the ionic strength was set at a constant level (0.01 M) by adding KCl. This eliminated the effect of ionic strength on the swelling equilibrium. The change in the gel volume, caused by the changes in either temperature or pH, was determined from the change in sample diameter by using an inverted optical microscope (Olympus, model PME 3) equipped with a calibrated scale. During the experiments, the temperature was controlled using a refrigerated circulator (Polysta, Cole Parmer). For a given temperature and pH, the swelling ratio for the rod-shaped gels was defined as V/Vo ) (l/lo)(d/do)2, where V and Vo represent the equilibrium volume of the hydrogel and the initial gel volume, d and l are the diameter and the length of the gel rod, and do and lo denote the diameter and the length of the capillary in which the gel was synthesized. Since the determination of l was troublesome (the ends of the gel rods after removal from capillaries and after cutting were not well-defined), the following approximation for the swelling ratio was taken in all measurements: V/Vo ) (d/do)3. The precision of the measurement of the diameter of the gel rods was better than 3%.
Results and Discussion Figure 3 shows the swelling ratio, (d/do)3, of NIPA-Z gels as a function of pH. The temperature of the measurement was 25 °C, which is well below the phase transformation temperature. The swelling ratio decreased rapidly in the pH region from 2 to 4. Then, in the region 4 < pH < 9, the dependence was fairly stable. The swelling ratio started again to rise strongly at pH 9. The changes seen in Figure 3 can be related to the acid-base equilibrium of the amino acids built into the polymer chains. Three amino acid species exist in the gels: I (cation, protonated amino group), II neutral form (dissociated carboxylic groups and protonated amino group, zwitterions), and III (anion, dissociated carboxylic group). The molar fractions (XI, XII, and XIII) of each form of the amino acid can be calculated using the following equations:
K a1 Ka1Ka2 1 )1+ + + + 2 XI [H ] [H ] Ka2 [H+] 1 ) +1+ + XII K a1 [H ] + 2
+
[H ] [H ] 1 ) + +1 XIII Ka1Ka2 Ka2
Figure 4. Calculated distribution of amino acid groups in function of pH. pKa1 ) 2.36 and pKa2 ) 9.60.
The previous equation is taken from the D. Skoog, D. West, and F. Holler analytical chemistry textbook.31 The values of Ka1 and Ka2 needed for the calculations were taken as those for the free aliphatic amino acid (leucine): pKa1 ) 2.36 and pKa1 ) 9.60.27 The plots shown in Figure 3 exhibit a minimum characteristic for ampholytic polymer networks.28,29 The pH range over which the minimum in Figure 3 is spread corresponds well to the pH distribution of the XII form shown in Figure 4. In this range, almost all amino acid groups are present as the zwitterions. Here, the van der Waals and hydrophobic interactions contribute significantly to the collapse of the ampholytic polymer networks. Moreover, the presence of the zwitterions and the practical absence of the XI and XIII forms decrease the osmotic pressure. Additionally, in this region, both the electrostatic repulsions between the groups similarly charged and the Coulombic attractions between the positive and negative charges (the number of which is equal) exist. These could lead to the collapse as well. At low and high pH, the behavior of the ampholytic networks is dominated by that corresponding to the ionized forms (XI or XIII). The ionized groups create an osmotic pressure in the network and therefore prompt the swelling process. Also, the charges on the ionized groups generate electrostatic repulsive forces between the polymer chains, which lead to further swelling of the network. We tried to compare our results with the results obtained for the gels containing separate acidic and basic groups. In ref 28, such a gel is described. That gel containes NIPA, acrylic acid, 1-vinylimidazole, and BIS in the molar ratio 69:15:15:1, respectively. A comparison of the data indicates that the swelling ratio in ref 28 in the minimum region is smaller. By analyzing the data in Figure 3 of this paper, we can arrive at a conclusion that if the content of zwitterions is increased, the swelling ratio for the minimum should further increase. We believe the discussed differences can be explained by easier (and therefore stronger) attractions among the independent ions in the Kokufuta gels. In our case (zwitterions), the opposite ions are closely located on the same monomer, and this may make the attraction process more difficult. (27) Handbook of Chemistry and Physics, 78th ed.; Lide, D. R., Ed.; CRC Press: New York, 1997. (28) Kokufuta, E. Langmuir 2005, 21, 10004. (29) Demosthenous, E.; Hadjiyannakou, S. C.; Vamvakaki, M.; Patrickios, C. S. Macromolecules 2002, 35, 2252.
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Figure 5. Temperature dependence of the degree of swelling for NIPA-Z gels with various compositions (y ) 0, 0.5, 1, 2, and 4%) at various pH values.
The volume phase transition with respect to T and y (percentage of the amino acid) is presented for three selected pH values in Figure 5. The nonionic NIPA gel (y ) 0%) shows a discontinuous volume phase transition at about 34 °C and, if the ionic strength is kept constant, this temperature does not change significantly over a pH range of 2-12. In Figure 5a,c the results obtained for pH 2.0 and 12.0 are shown. The volume change behavior of NIPA-Z gel changes from the discontinuous type to the continuous one as the amino acid group content increases. In these cases, a significant number of the amino acid groups on the polymeric chains are charged (at pH 2.0 about 40% and at pH 12.0 almost 100%, according to the data presented in Figure 4). The previous production of charge in the polymer network and its effect on the volume phase transition is similar to that reported for the gels with built-in either acidic or basic groups.21,30
Figure 6. Three-dimensional diagram of equilibrium swelling as functions of pH and temperature for NIPA-Z gels. (a) 0, (b) 2, and (c) 4%.
In Figure 5b, the results obtained for pH 6.1 are shown. In this case, contrary to Figure 5a,c, for y in the range of 0-4%, the identical (d/d0)3 value of 0.096 (for the completely collapsed state) is obtained in the phase transformation processes. For this pH, the shrinking process is the most advanced. The pH value of 6.1 is very close to the isoelectric point where almost all amino acid groups exist as the internal salts (the experimentally estimated isoelectric point, based on the 4% data in Figure 3 and taken as the central point in the wide minimum, is ca. 6.4). Thus, the attractions between the simultaneously positively and negatively charged groups on the polymer chains and the decrease
N-Isopropylacrylamide Networks Incorporating L-Lysine
in the osmotic pressure lead to more efficient collapses of the networks. For further characterization of the swelling behavior of the gels associated with a possibility of setting the appropriate charge excess and charge sign, two gels were selected with an amino acid content of 2% (Figure 6b) and 4% (Figure 6c). As a reference point, the plot for the gel with no amino acid is also given (see Figure 6a). The temperature dependence of the swelling ratio was examined at seven selected values of pH and is represented in a three-dimensional diagram in Figure 6. In both cases (Figure 6b,c), the surfaces presented are qualitatively similar. The changes in the swelling ratio are not very significant when the temperature is lower than 37 °C; the surfaces are almost flat. The situation is different for 2% NIPA-Z in a range of temperatures from 37 to 42 °C. Here, temperature and pH strongly influence the swelling behavior. For temperatures above 42 °C, in the region of the pH where the zwitterions dominate, the surface is also nearly flat, which indicates that the gel achieves a completely collapsed state. At the pH extremes, the surface is distinctly curved. The observed influence of pH on the swelling ratio is stronger in the case of 4% NIPA-Z. At temperatures above 37 °C, the curvature of the surface is sound, and it can be interpreted in terms of an incomplete collapse (in the investigated range of temperature, 20-50 °C). For the gel with no amino acid, the pH has negligible influence on the swelling behavior, and the temperature triggered volume change is always very abrupt.
Conclusions The new ampholytic polymer gels (NIPA-Z) based on N-isopropylacrylamide (NIPA) and modified natural amino acid L-lysine (Z) have been synthesized. The swelling properties of (30) Kawasaki, H.; Sasaki, S.; Maeda, H. Langmuir 2000, 16, 3195. (31) Skoog, D.; West, D.; Holler, F. Analytical Chemistry.
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the NIPA-Z gels have been studied. It was found that the swelling behavior of a hydrogel depends on the percentage of Z, pH, and temperature of the surrounding medium. The amino acids can exist in the gels in three forms: as cations, as a net neutral form (zwitterions), and as anions, which gives the possibility of controlling the excessive charge on the polymeric network. In the pH range, where zwitterions dominate, the osmotic pressure is low, and the interactions between the chains lead to the shrinking of the polymer network as well. According to the presented results and calculations, the zwitterions dominate in a quite wide range of pH from 4 to 9. The new gels give a chance of working with an excess of either positive or negative charge. The net charge close to zero is also obtainable in a rather wide range of pH. Such a situation widens the possibilities of using the shrinking process in different temperatures and pH. As was recently reported by our group,2 there is a possibility of anchoring a thin layer of the NIPA gel on the surface of the electrodes and exploiting the shrinking process at the surface. The addition of the amino acid group to the polymer chain may widen the possibilities at the electrode surface. It might be interesting to examine the transport of ions in this type of film and to construct matrixes for electrocatalysts at the electrodes. It is known that the pH of physiological liquids changes from 1 to 3 in the stomach to around 7 in the small and large intestine.27 Interestingly the 2% gel exhibits the highest sensitivity to pH changes at 37 °C. This provides more potential possibilities in medical applications. Acknowledgment. This work was financially supported by Ministry of Scientific Research and Information Technology under Project PBZ 18-KBN-098/T09/2003. LA060334N
