3338
Langmuir 1993,9, 3338-3340
Surfactant Effect on the Inverse Volume Phase Transition of a Polymer with Amino Acid Side Chains A. Safranj,*9+M. Yoshida,? H. Omichi,? and R. Katakaix Department of Material Development, Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, 1233 Watanuki, Takasaki, Gunma 370-12, Japan, and Department of Chemistry, Faculty of Engineering, Gunma University, Kiryu, Gunma 376, Japan Received August 2, 1993. In Final Form: October 1 9 , 1 9 9 9 We have synthesized poly(acryloy1-L-prolinemethyl ester) polymers and loosely-cross-linkedgels which demonstrate inverse volume phase transition in aqueous solutions. We studied the effect of increasing hydrophobicity of the L-proline side chain on the phase transition of the polymer in the presence of surfactant molecules. In view of the surfactant effect, we propose a method for determination of the phase transition temperature of LCST polymers. This method is especially valuable for those polymers having LCST lower than 0 OC, which is the case for many proteins. Polymers which exhibit good solubility in aqueous solutions at low temperatures, but separate from the solvent when temperature is raised above a certain level called the lower critical solution temperature (LCST),have received much interest in recent years, motivated by theoretical, biomedical, and industrial concerns. Crosslinked polymers which show LCST behavior have been proposed for various applications ranging from devices for controlled drug releasel to enzyme reactors2and solute ~eparation.~ One important features of these systems is that the LCST of the polymer can easily be controlled by varying the number of hydrophobic, hydrophilic, or charged groups on the polymer or by additives, such as inorganic salts or surfactants to the solutions. Many recent studies deal with the effect of hydrophobicity, ionization, and additives on the phase transition of poly(N-isopropylacrylamide),the best-known and extensively studied LCST polymer.P10 This polymer is interesting not only because of many applications in various areas but also as a model for understanding the folding and unfolding of biopolymers, the process that affects many essential functions. At low temperatures, the peptide chains of biopolymers are in fully hydrated, unfolded state, but with increasingtemperature toa critical point, the polypeptide chains fold into a functional state. (The reverse process is the so-called cold denaturation.) With a further increase in the temperature, thermal denaturation takes place. The understanding of this mechanism in detail is difficult in real systems, due to the 'Japan Atomic Energy Research Institute. t Gunma University. Abstract published in Aduance ACS Abstracts, December 1, @
1993. (1)Hoffman, A. S.; Afrassiabi, A.; Dona, L. C. J. Controlled Release 1986,4, 213. (2) Park, T. G.; Hoffman, A. S. Appl. Biochem. Biotechnol. 1988,19, 1.
(3) Feil, H.; Bae, Y. H.; Feijen, J.; Kim, S. W. J. Membr. Sci. 1991,64, 283. (4) Ringsdorf, H.; Venzmer, 3.;Winnik, F. M. Macromolecules 1991, 24, 1678. ( 5 ) Beltran, S.; Baker, J. P.; Hooper, H. H.; Blanch, H. B.; Prausnitz, J. M. Macromolecules 1991,24, 549. (6) Hirotau, S.; Hirokawa, Y.; Tanaka, T. J. Chem. Phys. 1987,97 (2), 1392. (7) Feil, H.; Bae, Y.-H.; Feijen, J.; Kim, S. W. Macromolecules 1993, 26, 2496. (8)Schild, H. G.; Tirrell, D. A. Langmuir 1990,6, 1676. (9) Wada, N.; Kajima, Y.; Yagi, Y.; Inomata, H.; Saito, S. Langmuir 1993, 9, 46.
(10)Kokufuta, E.; Zhang, Y.-Q.; Tanaka, T.; Mamada, A. Macromolecules 1993,26, 1053.
/
n
R= CH, CHzCH3 CH2 CHzCH 3
Figure 1. Structural formula for A-ProOR. Detailed experimental procedure of the synthesis is published in our previous
paper.I4
complexity of biological macromolecules. An acceptable model system for proteins, besides the N-isopropylacrylamide could be a natural sequence from a protein, a peptide, or amino acid based polymer. Studies of the LCST behavior of elastin-based polypeptides have been recently published.11J2 We were interested in using a-amino acids and have synthesized loosely cross-linked poly(acryloy1-L-proline methyl ester) hydrogelsl3 and hydrophilic p01ymers.l~ These polymers have L-proline as a pendant group and show LCST behavior, and the gels could be used as drug delivery systems.lsJ5 Here we summarized the effects of the increasing hydrophobicity of this proline side chain on the LCST of the polymer and the interaction between the polymers and surfactant molecules added to the solution. In view of the surfactant effect we propose a method for determination of the phase transition temperature of LCST polymers. The interaction of several different surfactants (anionic, cationic, zwitterionic, and nonionic) with our gels will be published later in full detail. Acryloyl-L-proline alkyl esters (A-ProOR, where R = Me, Et, and Pr are methyl, ethyl, and propyl groups, respectively) were synthesized by a coupling reaction (11) Luan,C.-H.;Parker,T.M.;Prasad,K.U.;Urry,D. W.Biopolymers 1991, 31, 465. (12) Luan, C.-H.; Urry,D. W. J. Phys. Chem. 1991,95,7896.
(13) Yoshida, M.; Asano, M.; Kumakura, M.; Katakai, R.; Mashimo, T.; Yuasa, H.; Yamanaka, H. Drug Des. Delivery 1991, 7, 159. (14) Yoshida, M.; Omichi, H.; Katakai, K. Eur. Polym. J. 1992,28 (9), 1141. (15) Miyajima, M.; Yoshida, M.; Sato, H.; Omichi, H.; Katakai, R.; Higuchi, W. I. Int. J.Pharm. 1993, 95, 153.
0743-7463/93/2409-3338$04.00/00 1993 American Chemical Society
Letters
Langmuir, Vol. 9,No. 12, 1993 3339
8I -10
0
10
20
Mixed micelle
I
30
Temperature ("C)
Figure 2. DSC curves of poly(A-ProOMe) (a),poly(A-ProOEt) (b),and poly(A-ProOPr)(c), in 10% (w/v)aqueous solution.The maximum of the endotherm(pointedat by the arrows)represents the cloud point. These measurements were done by using a Seiko DSC-10 differential scanning calorimeter, with heating rate of 1deg/min.
1
Sudictant
Extended coil
MiceIIe
I
Shrunken coil (cluster!
reported in detail e1~ewhere.l~ The monomers (chemical structure shown in Figure 1)were polymerized by radiation (30kGy) at a dose rate of 10kGy/h, at 25 "C, under nitrogen atmosphere. The resulting homopolymers were precipitated from water, lyophilized and used for preparation of solutions. The LCST temperatures of poly(A-ProOR) were determined by cloud point and DSC measurements in the range of 1-1093 (w/v) polymer concentration. The cloud points correspond to the temperatures at the maximum of the DSC endotherm (Figure 2). Poly(AProOMe) and poly(A-ProOEt) show phase separation at i4 and 2 "C, respectively,whilepoly(A-ProOPr) is insoluble in water at all tempertitures. It has been suggested that the LCST behavior is caused by critical balance of hydrophobic and hydrophilic groups in polymer side chain.l6 In our systems, CH2 and CH3 groups in the polymer backbone and in the proline side chain are the hydrophobic component and the C=O and N the hydrophilic moieties. Below the LCST, poly(A-ProOMe) shows the extended coil structure with strong interaction between = 40 ProOMe groups and water (radius of gyration (S)1/2 nm, and apparent volume of 270 000 nm3 at 10 "C, as we have determined from light scattering studies).14 With increasing temperature, the hydrophobic interactions between proline groups become dominant and, a t temperatures above the LCST, lead to inter- and intramolecular interactions forming compact clusters (radius of gyration (S)l/z= 8 nm, apparent volume of 2100 nm3 at 50 "C).14 At low temperatures the strong hydrogen bonding between hydrophilic groups and water triggers the formation of a highly organized water layer around the polymer chains. The formation of this structured water contributes favorably to the enthalpy of mixing, which outweighs the unfavorable free energy related to the exposure of hydrophobic groups to water thus enabling the good solubility of polymer. With increasing temperature, hydrogen bonding weakens, the structured water is released, and the interactions between hydrophobic side groups increase. Above the LCST, these hydrophobic interactions become dominant (the free energy of mixing takes a positive value) and lead to an entropy-driven collapse of polymer chains from an expanded coil to a compact globular conformation and phase separation.l'J8 The model for this phase transition is shown in Figure 3, lower part.
(17) Bae, Y. H.; Okano, T.; Kim, S. W. J. Polym. Sci., Polym. Phys. Ed. 1990,28,923. (18) Otake, K.; Inomata, H.; Konno, M.; Saito, S. Macromolecules
(16) Taylor, L. D.; Cerankowaki, L. D. J. Polym. Sci., Polym. Chem. Ed. 1975, 13, 2551.
1990, 23, 283. (19) Inomata, H.; Goto, S.;Otake, K.; Saito,S. Langmuir 1992,8,687. (20) Schild, H. G.; Tirrell, D. A. Langmuir 1991, 7,665.
LCSTo
LCSTI LCSTz
Temperature
Figure 3. Proposed model for the phase transition of poly(acryloyl-L-prolinealkyl esters). In aqueous solution (lower part of the diagram), at temperatures below LCSTo, the polymers have extended coil structure with strong interactions between the ProOR groups and water. A t higher temperatures, this interaction weakens, and the probability for inter- and intramolecular interactions between the neighboring proline groups increases. At temperaturesabove the LCSTo, those interactions will lead to formation of big flat clusters and phase separation. When surfactant is present in the solution, the surfactant molecules will aggregate on the hydrophobic proline side chain formingmixed micelles (upperpart of the diagram). The charged head groups of the surfactants in those micelles will act as a barrier between the proline groups thus increasing the temperaturefor phase separationto LCST1. Further surfactantaddition will increase the size of alreadyformed mixed micelles, increasing even further the phase transition temperature to LCST2. In their classic study,l6 Taylor and Cerankowski proposed as a general rule that the LCST of water-soluble polymers should decrease with increasing hydrophobicity of the polymer. Our results clearly show that the transformation temperature is much affected by a small increase of the hydrophobicity of the proline side chains, changing it from methyl to propyl, underlining further the important role of hydrophobic interactions in this system. If we assume that addition of one CH2 unit to the polymer side chain lowers the LCST by about 12 "C, as our results seem to indicate, we would expect poly(A-ProOPr)to have LCST at around -10 "C. This presumption however, could not be proved in pure aqueous systems. The swellingproperties of gels and transition properties of polymers can be also controlled by the use of additives, such as inorganic salts and surfactants. It has been shown that addition of inorganic salts to a nonionic polymer solution usually lowers the LCST of that polymer, and this change depends on the anion rather than on the cation.lg If a surfactant is added to the polymer solution, the interaction will depend strongly on structural parameters, such as the charge of the surfactant and the length of its tail.20 Generally, nonionic polymers tend to form
3340 Langmuir, Vol. 9, No. 12,1993
r
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--
0
4
8
12
16
200
SDS concentration
100 200 300 400 500 (mM)
Figure 4. Cloud points of poly(A-ProOR) in aqueous solution as a function of the concentrationof added surfactant: (a) poly(A-ProOMe);(b)poly(A-ProOEt);(c)poly(A-ProOPr). Note that the LCST temperatures obtained by extrapolation to zero SDS concentrationagree well with those determined by DSC and cloud point measurement.
complexes with ionic surfactants and these complexes behave like polyelectrolytes.21 Most nonionic watersoluble polymers owe this solubility to their polar groups, such as hydroxyl, carboxyl, or amide, which hydrates in water. This hydration diminishes with temperature (as discussed above) and the polymer precipitates. When a charged surfactant aggregates on the polymer, it creates an electrostatic barrier which opposes the collapse of the polymer, thus enhancing its solubility. One would expect therefore the elevation of the LCST in such systems. We have investigated the interaction between poly(acryloyl-L-prolinealkyl esters) and an anionic surfactants, sodium dodecyl sulfate (SDS). Figure 4a shows the observed changes in transition temperature as a function of the SDS concentration in the solutions of A-ProOMe and A-ProOEt. As expected for the system consisting of (21) Goddard, E. G. Colloids Surf. 1986,19,255.
Letters
neutral water-soluble polymer and charged surfactant,21 the binding of the surfactant to the polymer starts at concentrations somewhat below the critical micelle concentration (8 mM for SDS). This binding is cooperative: 22 further addition of surfactant results in large increase in the amount of bound surfactant, while the amount of free surfactant remains almost constant. The relationship between the LCST and the surfactant concentration is linear for poly(A-ProOR) (Figure 4a). When extrapolated to zero SDS concentration, the temperatures obtained in this way for both poly(A-ProOMe) and poly(A-ProOEt) agree well with the ones from DSC and cloud point measurements. SDS solubilizes poly(AProOPr), and we can determine its phase transition temperature. Our method of extrapolation gives the LCST for poly(A-ProOPr) around -11 "C (Figure 4b), which is near the predicted one. In analogy with the hydrophobically modified NIPAMSDS we propose a model for our system, shown in Figure 3 (upper part). The surfactants bind preferentially to the hydrophobic sites on the proline side chain thus forming mixed micelles or clusters, and with the increase of the surfactant concentration those micelles grow only in size but not in number. The electrostatic barrier which is created in this way around the proline side chain prevents them from hydrophobic interactions and, thus, the separation of the polymer from the solution. This phenomenon will lead to the elevation of the phase transition temperature. We believe that the method described here can be used successfully in other similar cases, especially for those proteins which have their cold denaturation temperature below the freezing point of aqueous solutions. (22) Winnik, F. M.;Ringsdorf, H.; Venzmer,J. Longmuir 1991,7,912.
