Langmuir 1998, 14, 6059-6067
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Hydrophobic Self-Association of Cholesterol Moieties Covalently Linked to Polyelectrolytes: Effect of Spacer Bond Shin-ichi Yusa,† Mikiharu Kamachi, and Yotaro Morishima* Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan Received December 18, 1997. In Final Form: July 21, 1998 Polyelectrolytes carrying cholesterol (Chol) pendants were synthesized by free-radical copolymerization of sodium 2-(acrylamido)-2-methylpropanesulfonate with cholesteryl methacrylate (CholMA) or cholesteryl 6-methacryloyloxyhexanoate (Chol-C5-MA), with the contents of CholMA and Chol-C5-MA units in the copolymers ranging from 0.5 to 1.0 and 0.5 to 10 mol %, respectively. In the CholMA copolymer, Chol is directly linked to the polymer main chain by an ester bond, whereas in the Chol-C5-MA copolymer, Chol is linked via a pentamethylene spacer. The association behavior of these Chol-carrying polymers in aqueous solution was investigated by turbidimetry, 1H NMR, fluorescence, size exclusion chromatography (SEC), static light scattering (SLS), quasielastic light scattering (QELS), and viscometry, with a focus on the effect of the spacer bond on the self-association of Chol pendants. For fluorescence studies, Chol-carrying polymers labeled with a pyrene (Py) or naphthalene (Np) moiety were employed. Fluorescence of molecular Py solubilized in the hydrophobic microdomains of the polymers was also examined. Pyrene fluorescence data indicated that the Chol pendants underwent self-association in aqueous solution even if their contents in the polymers were as low as 1 mol %. For the 5 mol % Chol-C5-MA-containing polymer, a strong tendency for interpolymer association was indicated by nonradiative energy transfer from Np to Py chromophores labeled on separate polymer chains. Proton NMR data for the 5 mol % Chol-C5-MA-containing polymer in D2O indicated highly restricted motions of Chol groups arising from the self-association of Chol groups. Taken together with SEC data, SLS and QELS data showed that the Chol-C5-MA copolymers have a much stronger propensity for interpolymer association than the CholMA copolymers, with the pentamethylene spacer bond facilitating pendant Chol groups to undergo interpolymer association. On the basis of the characterization data, an intermolecularly bridged “flower-type” micelle model was proposed for the aggregates of the Chol-C5-MA copolymers.
Introduction Over the past decade, the self-organization of hydrophobically modified water-soluble polymers has been a focus of interest from both scientific and practical perspectives.1-12 In aqueous solution, association of hydrophobes covalently linked to water-soluble polymers can occur intra- or intermolecularly. Interpolymer association would lead to bulk phase separation, such as gelation and precipitation, whereas intrapolymer association leads to the formation of unimolecular micelles (unimer micelles). In highly dilute solutions, in general, intrapolymer association may preferably occur, whereas in concentrated solutions, interpolymer association is * To whom correspondence should be addressed. † Present address: Department of Applied Chemistry, Himeji Institute of Technology, Shosha, Himeji 671-2201, Japan. (1) Zhang, Y. X.; Da, A. H.; Hogen-Esch, T. E.; Butler, G. B. In Water Soluble Polymers: Synthesis, Solution Properties and Application; Shalaby, S. W., McCormick, C. L., Butler, G. B., Eds.; ACS Symposium Series 467; America Chemical Society: Washington, DC, 1991; p 159. (2) Schmolka, I. R. J. Am. Oil. Chem. Soc. 1991, 68, 206. (3) Almgren, M.; Bahadur, P.; Jansson, M.; Li, P.; Brown, W.; Bahadur, A. J. Colloid Interface Sci. 1991, 151, 157. (4) Malmsten, M.; Lindman, B. Macromolecules 1992, 25, 5440. (5) Linse, P.; Bjo¨rling, M. Macromolecules 1991, 24, 6700. (6) Linse, P.; Malmsten, M. Macromolecules 1992, 25, 5434. (7) Linse, P. J. Phys. Chem. 1993, 97, 13896. (8) Hurter, P. N.; Scheutjens, J. M. H. M.; Hatton, A. T. Macromolecules 1993, 26, 5592. (9) Malmsten, M.; Linse, P.; Zhang, K.-W. Macromolecules 1993, 26, 2905. (10) Glatter, O.; Gu¨nther, S.; Schille´n, K.; Brown, W. Macromolecules 1994, 27, 6046. (11) Linse, P. Macromolecules 1993, 26, 4437. (12) Linse, P. Macromolecules 1994, 27, 2685.
likely to occur. However, our recent studies showed that the preference of intra- or interpolymer hydrophobic selfassociation depends on the chemical structure of the polymer. We reported that copolymers of sodium 2(acrylamido)-2-methylpropanesulfonate (AMPS) and a methacrylamide N-substituted with such a bulky hydrophobic group as cyclododecyl, adamantyl, or naphthylmethyl group undergo predominant intrapolymer hydrophobic self-association in aqueous solution, forming unimer micelles regardless of the polymer concentration.13-18 In these copolymers, bulky hydrophobes are connected to the polymer backbone via amide bonds and the electrolyte and hydrophobic monomer units are randomly distributed along the main chain. It appears that these structural features are determinant factors for predominant intrapolymer association. In fact, McCormick et al.19 showed that blocky sequences of hydrophobic monomer units in hydrophobically modified water-soluble copolymers have a tendency for interpolymer association, whereas random sequences tend to associate intramolecularly. (13) Morishima, Y. Prog. Polym. Sci. 1990, 15, 949. (14) Morishima, Y. Adv. Polym. Sci. 1992, 104, 51. (15) Morishima, Y. Trends Polym. Sci. 1994, 2, 31. (16) Morishima, Y. Bio-Industry 1995, 12, 20. (17) Morishima, Y.; Seki, M.; Nomura, S.; Kamachi, M. In Macroion Characterization: From Dilute Solutions to Complex Fluids; Schmitz, K. S., Ed.; ACS Symposium Series 548; America Chemical Society: Washington, DC, 1994; p 243. (18) Morishima, Y. In Multidimensional Spectroscopy of Polymers: Vibrational, NMR, and Fluorescence Techniques; Urban, M. W., Provder, T., Eds.; ACS Symposium Series 598; American Chemical Society: Washington, DC, 1995; p 490. (19) Chang, Y.; McCormick, C. L. Macromolecules 1993, 26, 6121.
S0743-7463(97)01388-7 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/19/1998
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In hydrophobically modified polyelectrolytes, in general, hydrophobic association occurs in competition with electrostatic repulsion. Furthermore, polymer chain is likely to exert steric constraints to hydrophobic self-association. Therefore, one may anticipate that the spacing between hydrophobes and the polymer backbone, or the motional and geometrical freedom of hydrophobes, is a key factor to determine whether intra- or interpolymer self-association of hydrophobes preferentially occurs. To investigate the effect of polymethylene spacers between hydrophobes and the polymer backbone, it should be important to choose an appropriate hydrophobe that shows a strong tendency for self-association as compared with associating tendency of the spacer groups. Akiyoshi and Sunamoto20-24 reported that pullulan, a polysaccharide, covalently modified with a few cholesterol (Chol) groups per 100 glucose units formed stable nanoparticles with diameters of 20-30 nm in water via interpolymer self-association of Chol groups. These results suggest that Chol groups have a strong propensity for self-association even if their contents in polymers are very low. These studies motivated us to employ Chol groups as a hydrophobe to investigate the effect of the spacer on the selfassociation of the hydrophobes linked to polyelectrolytes. In this study, we covalently incorporated small mole percents of Chol moieties into poly(AMPS). We synthesized two types of polymers (Chart 1); copolymers of AMPS with cholesteryl methacrylate (CholMA) and with cholesteryl 6-methacryloyloxyhexanoate (Chol-C5-MA). In the former, Chol is directly attached to the main chain by an ester bond, whereas in the latter, Chol is linked to the main chain via a pentamethylene spacer in connection with two ester bonds. The associating behavior of these polymers in aqueous solution was investigated by turbidimetry, 1H NMR, fluorescence, size exclusion chromatography (SEC), static light scattering (SLS), quasielastic light scattering (QELS), and viscometry. For fluorescence studies, terpolymers of AMPS, CholMA, or Chol-C5-MA, and N-(1-pyrenylmethyl)methacrylamide or N-(1-naphthylmethyl)methacrylamide were employed (Chart 1).
Yusa et al. Chart 1. Chemical Structures of Polymers Studied
Experimental Section Monomers. 2-(Acrylamido)-2-methylpropanesulfonic acid (AMPS) was used as received from Nitto Chemical Industry Company. Cholesteryl 6-methacryloyloxyhexanoate (Chol-C5MA) was prepared according to the method reported by Shannon.25 Cholesteryl methacrylate (CholMA),26 N-(1-pyrenylmethyl)methacrylamide (1PyMAm),27 and N-(1-naphthylmethyl)methacrylamide (1NpMAm)28 were prepared as reported previously. Polymers. The terpolymers of AMPS, 1PyMAm, and CholMA (or Chol-C5-MA) were prepared by free-radical polymerization initiated by 2,2′-azobis(isobutyronitrile) (AIBN) in N,N-dimethylformamide (DMF). A procedure for the terpolymerization is as follows. A predetermined amount of AMPS was neutralized by equimolar Na2CO3 in a DMF solution. To this solution were (20) Akiyoshi, K.; Nagai, K.; Nishikawa, T.; Sunamoto, J. Chem. Lett. 1992, 1727. (21) Akiyoshi, K.; Deguchi, S.; Moriguchi, N.; Yamaguchi, S.; Sunamoto, J. Macromolecules 1993, 26, 3062. (22) Deguchi, S.; Akiyoshi, K.; Sunamoto, J. Macromol. Rapid Commun. 1994, 15, 705. (23) Nishikawa, T.; Akiyoshi, K.; Sunamoto, J. Macromolecules 1994, 27, 7654. (24) Akiyoshi, K.; Deguchi, S.; Tajimi, H.; Nishikawa, T.; Sunamoto, J. Proc. Jpn. Acad. 1995, 71, Ser. B, 15. (25) Shannon, P. J. Macromolecules 1983, 16, 1677. (26) de Visser, A. C.; de Groot, K.; Feyen, J.; Bantjes, A. J. Polym. Sci., Polym. Lett. Ed. 1972, 10, 851. (27) Morishima, Y.; Tominaga, Y.; Kamachi, M.; Okada, T.; Hirata, Y.; Mataga, N. J. Phys. Chem. 1991, 95, 6027. (28) Morishima, Y.; Tominaga, Y.; Nomura, S.; Kamachi, M. Macromolecules 1992, 25, 861.
added predetermined amounts of 1PyMAm, CholMA (or CholC5-MA), and AIBN. The solution was placed in a glass ampule and deaerated on a vacuum line by six freeze-pump-thaw cycles, and then the ampule was sealed under reduced pressure. Polymerization was carried out at 60 °C for 12 h. The polymerization mixture was poured into a large excess of ether to precipitate resulting polymers. The polymer was purified by reprecipitating from methanol into a large excess of ether three times. The polymer was then dissolved in pure water, and the aqueous solution was dialyzed against pure water for 1 week. The polymer was recovered by freeze-drying. The compositions of the terpolymers were determined by elemental analysis (C/N
Association of Polyelectrolyte-Bound Cholesterol ratio) and ultraviolet-visible (UV-visible) absorption spectroscopy. Because the solubility of CholMA monomer in DMF is so low, we prepared only copolymers of low CholMA contents (e1 mol %). The naphthalene (Np)-labeled polymer was prepared in a similar manner. The polymers are coded as poly(A/Chol-C5-MAx/Py), poly(A/ CholMAx/Py), and poly(A/Chol-C5-MAx/Np), where x represents the mol % content of Chol-C5-MA or CholMA in the copolymers. A homopolymer of AMPS and a copolymer of AMPS and 1 mol % 1PyMAm were prepared as described previously.27 Other Materials. Pyrene (Py) was purchased from Nacalai Tesque, Inc. and recrystallized from ethanol. Water was distilled and deionized by passing through an ion-exchange column. Measurements. (a) Turbidimetry. Turbidities of aqueous solutions of varying concentrations of the polymers were recorded as 100 - % transmittance (T) on a Shimadzu UV-2500PC spectrophotometer with a 1.0-cm path length quartz cell at 488 nm. Sample solutions were kept stirring during the measurement in the spectrophotometer, and % T values were recorded several minutes after their readings are stabilized. (b) NMR. Proton NMR spectra were obtained with a JEOL EX-270 or JEOL GSX-400 spectrometer using a deuterium lock at a constant temperature of 30 °C during the whole run. The NMR tubes containing D2O solutions of the polymers (20 mg/ mL) were deaerated by purging with Ar for 30 min. Proton spinlattice relaxation times (T1) were determined by an inversionrecovery technique with a 180°-τ-90° pulse sequence.29-31 Proton spin-spin relaxation times (T2) were determined by the Carr-Purcell-Meiboom-Gill (CPMG) method.32 (c) Absorption and Fluorescence Spectra. Absorption spectra were recorded on a Shimadzu UV-2500PC spectrophotometer. Fluorescence spectra were recorded on a Hitachi F-4500 fluorescence spectrometer with excitation at 343 nm for Py at room temperature. Excitation and emission slit widths were maintained at 5.0 and 2.5 nm, respectively. Measurements of fluorescence spectra of molecular Py solubilized in the polymers in water were carried out as follows. A 100-mL aliquot of acetone solution containing 10 mg of molecular Py was evaporated in a round-bottomed flask on a rotary evaporator to dryness in the form of a thin film. This evaporation was followed by the addition of 5 mL of a polymer solution (10 mg/mL) in pure water. The aqueous solution was mechanically stirred for 30 min and then sonicated for 30 min. To remove excess Py microcrystals, the solution was filtered with a 0.2 µm (pore size) poly(tetrafluoroethylene) (PTFE) filter prior to fluorescence measurements. (d) Fluorescence Decays. Fluorescence lifetimes for Py were measured with a Horiba NAES-550 system equipped with a flash lamp filled with hydrogen. Sample solutions were excited at 343 nm. The detection wavelength was set at 400 nm with a band-pass filter (Toshiba KL-40). Sample solutions for fluorescence lifetime measurements were purged with Ar for 30 min. (e) Nonradiative Energy Transfer (NRET). Interpolymer NRET experiments were performed as reported previously.33 A 0.0072 mg/mL aqueous solution of poly(A/Chol-C5-MA5/Py) was mixed with the same volume of a 0.0324 mg/mL aqueous solution of poly(A/Chol-C5-MA5/Np). To this mixed solution was added an aqueous solution of poly(A/Chol-C5-MA5) (nonlabeled polymer) such that the concentrations of the total polymers were varied in the range 0.02-2.3 mg/mL while the concentrations of the Py and Np labels were kept constant. The solutions were weakly sonicated for 5 min. The solutions of varying concentrations of the total polymers were subjected to fluorescence measurements. Fluorescence spectra were recorded on a Hitachi F-4500 spectrofluorometer with excitation at 290 nm at room temperature. Excitation and emission slit widths were maintained at 10.0 and 5.0 nm, respectively. Contribution from direct excitation of Py was corrected by subtracting from each spectrum the emission (29) Erdmann, K.; Gutsze, A. Colloid Polym. Sci. 1987, 265, 667. (30) Raby, P.; Budd, P. M.; Heatley, F.; Price, C. J. Polym. Sci., Polym. Phys. Ed. 1991, 29, 451. (31) Brereton, M. G.; Ward, I. M.; Boden, N.; Wright, P. Macromolecules 1991, 24, 2068. (32) Meiboom, S.; Gill, D. Rev. Sci. Instrum. 1958, 29, 688. (33) Morishima, Y.; Nomura, S.; Ikeda, T.; Seki, M.; Kamachi, M. Macromolecules 1995, 28, 2874.
Langmuir, Vol. 14, No. 21, 1998 6061 spectrum of the Py-labeled polymer of the same Py concentration. The intensities of Np and Py fluorescence were estimated at 326 and 395 nm, respectively. (e) Size Exclusion Chromatography (SEC). Analysis was performed at 70 °C with an SEC equipped with an UV-8010 UV detector (Tosoh). A combined column of TSKgel G6000PWXL and G3000PWXL (Tosoh) was employed. For the eluent, 0.2 M phosphate buffer containing 50% (v/v) acetonitrile was used. Standard sodium polystyrenesulfonates were used to calibrate the molecular weight. (f) Static Light Scattering (SLS). Data were obtained at 25 °C with an Otsuka Electronics Photal DLS-700 light scattering spectrometer equipped with an Ar laser (output power ) 75 mW at λ ) 488 nm). For SLS measurements, values of dn/dc (refractive index increment against concentration) were determined with an Otsuka DRM-1020 differential refractometer. Sample solutions were filtered with 0.1 or 0.2 µm pore size membrane filters prior to measurement. (g) Quasielastic Light Scattering (QELS). Data were obtained at 25 °C with an Otsuka Electronics Photal DLS-700 light scattering spectrometer equipped with a multi-τ, digital time correlator (ALV-5000). A 488 nm Ar laser was used at light source and output power was 75 mW. Sample solutions of polymers in 0.1 M NaCl were filtered with 0.1 or 0.2 µm pore size membrane filters prior to measurement. The intensity autocorrelation function g(2)(t) was measured by QELS. The intensity autocorrelation function is related to the normalized autocorrelation function g(1)(t) as follows
g(2)(t) ) B[1 + β|g(1)(t)|2]
(1)
where β is a parameter of the optical system (constant) and B is a baseline. The inverse Laplace transform (ILT) analysis of the normalized intensity autocorrelation functions, g(2)(t), was performed using the algorithm REPES34 to obtain the relaxation time distribution, τA(τ), according to
g(1)(t) )
∫ τA(τ) exp(-t/τ) d ln τ
(2)
where g(1)(t) is the normalized first-order electric field time correlation function, τ is the relaxation time. The relaxation time distribution is presented as τA(τ) versus log τ profile, with τA(τ), providing an equal area representation. The details of QELS instrumentation and theory are described in the literature.35,36 (h) Viscometry. Measurements were carried out for all samples at 30 °C using a modified Ubbelohde type viscometer. Sample solutions were filtered with 0.2 µm pore size membrane filters prior to measurement.
Results and Discussion In the present study, two types of Chol-carrying polyanions were synthesized: they are, the copolymers of AMPS and Chol-C5-MA, where Chol is linked to the polymer backbone via a pentamethylene spacer bond; and the copolymers of AMPS and CholMA, where Chol is directly linked to the backbone by an ester bond. Copolymerizations of varying molar ratios of AMPS and CholC5-MA were performed in the presence of AIBN in DMF at 60 °C, and copolymers with Chol-C5-MA contents ranging from 0.5 to 10 mol % (Chart 1) were prepared. The copolymer compositions were close to monomer feed compositions (Figure 1). The monomer reactivity ratios for Chol-C5-MA (M1) and AMPS (M2) were r1 ) 1.14 ( 0.32 and r2 ) 0.91 ( 0.31, respectively, as determined by the best fit of the composition data to the copolymer composition equation. These results indicate that the distribution of the two monomer units in these copolymers is virtually random. On the other hand, copolymerizations of CholMA (34) Jakes, J. Czech. J. Phys. 1988, B38, 1305. (35) Phillies, G. D. J. Anal. Chem. 1990, 62, 1049A. (36) Phillies, G, D, J. J. Chem. Phys. 1988, 89, 91.
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Figure 1. Composition plot for the copolymerization of CholC5-MA (M1) and AMPS (M2) in DMF at 60 °C. A theoretical curve for the copolymerization composition equation with r1 ) 1.14 ( 0.32 and r2 ) 0.91 ( 0.31 is indicated.
Figure 3. The 270 MHz 1H NMR spectra of poly(A/Chol-C5MA5) in D2O and in DMSO-d6 at 30 °C.
Figure 2. Turbidity at 488 nm as a function of the polymer concentration in aqueous solution. Key: (O) poly(A/Py); (4) poly(A/CholMA0.5/Py); (0) poly(A/CholMA1/Py); (3) poly(A/CholC5-MA0.5/Py); ()) poly(A/Chol-C5-MA1/Py); (×) poly(A/CholC5-MA5/Py); (b) poly(A/Chol-C5-MA7/Py); (2) poly(A/Chol-C5MA10/Py).
and AMPS were only possible with CholMA 1 mg/mL. This increase in IPy/INp is due to a decrease in the average distance between the Np and Py labels; that is, with increasing polymer concentration, increasing fractions of Np and Py labels come close to each other within the Fo¨rster radius (R0 ) 2.86 nm for transfer from 1-methylnaphthalene to Py49). These results indicate that interpolymer selfassociation of Chol groups occurs even in a very low concentration regime (e0.9 mg/mL) and that interpolymer Chol association occurs extensively at concentrations >1 mg/mL. We attempted to estimate the molar masses of the polymers by SEC with a mixed solvent of water (0.2 M phosphate buffer) and acetonitrile (50/50, v/v) as the eluent. Weight-average molecular weights (Mw) estimated by SEC are listed in Table 3. The reference and all the Chol-containing polymers show similar values of Mw. Thus, it is inferred that interpolymer Chol association is absent in the water/acetonitrile (50/50, v/v) mixed solvent and that the molar masses for all the polymers are more or less the same. In contrast, Mw values for the Chol-C5(48) Kramer, M. C.; Steger, J. R.; Hu, Y.; McCormick, C. L. Macromolecules 1996, 29, 1992. (49) Berlman, I. B. Energy Transfer Parameters of Aromatic Compounds; Academic: New York, 1973.
Association of Polyelectrolyte-Bound Cholesterol
Figure 9. Zimm plots for (a) poly(A/CholMA1/Py) and (b) poly(A/Chol-C5-MA1/Py) in 0.1 M NaCl aqueous solution at 25 °C.
MA-containing polymers estimated by SLS in 0.1 M NaCl aqueous solution were much greater than those estimated by SEC in water/acetonitrile (50/50, v/v). Figure 9 compares Zimm plots for the CholMA- and Chol-C5-MAcontaining polymers, both the polymers containing 1 mol % Chol unit. The measurements of SLS were performed in a 0.5-4.0 mg/mL range of polymer concentrations. The Zimm plots for the Chol-C5-MA-containing polymer did not yield straight lines, arising from interpolymer association in this concentration range. The apparent Mw values were estimated by the extrapolation of θ to zero. The results for the reference and Chol-containing polymers with varying CholMA and Chol-C5-MA contents are summarized in Table 3. The apparent Mw values for the Chol-C5-MA-containing polymers are much larger than those estimated by SEC, and the apparent Mw values for the 0.5 and 1 mol % Chol-C5-MA polymers are much higher than those for the CholMA-containing polymer of the same Chol contents. In the Chol-C5-MA-containing polymers, the apparent Mw values markedly increase with increasing Chol content, arising from a marked increase in the extent of interpolymer association with increasing the Chol content. The apparent Mw values for the 0.5 and 1 mol % CholMA polymers are practically the same, and the Mw values are slightly higher than that of the reference polymer. The apparent mean-square radii of gyration (1/2), estimated from the slopes of the angular dependence in Zimm plots in a chosen θ range, are also listed in Table 3. The apparent 1/2 for the 1 mol % Chol-C5-MA polymer (50.3 nm) is much larger than that of the CholMA-containing polymer of the same Chol content (21.7 nm). Thus, there is a clear tendency that Mw and 1/2 for the Chol-C5-MA-containing polymers are considerably larger than those for the CholMAcontaining polymers of the same Chol contents. This result is an indication of interpolymer association of the polymers with Chol groups linked to the main chain via a pen-
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tamethylene spacer. On the other hand, no such interpolymer association occurs for the polymers without the spacer when Chol contents are
