Hybrid Polymeric Micelles with Hydrophobic Cores and Mixed

Jun 6, 2001 - Department of Physical Chemistry, Uppsala University, Box 532, 751 21 Uppsala, Sweden. Langmuir , 2001, 17 (14), pp 4245–4250...
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Langmuir 2001, 17, 4245-4250

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Hybrid Polymeric Micelles with Hydrophobic Cores and Mixed Polyelectrolyte/Nonelectrolyte Shells in Aqueous Media. 2. Studies of the Shell Behavior† Kla´ra Podha´jecka´, Miroslav Sˇ teˇpa´nek, and Karel Procha´zka* Department of Physical and Macromolecular Chemistry, School of Science, Charles University in Prague, and Laboratory of Specialty Polymers‡, Albertov 2030, 12840 Prague 2, Czech Republic

Wyn Brown Department of Physical Chemistry, Uppsala University, Box 532, 751 21 Uppsala, Sweden Received February 16, 2001. In Final Form: April 11, 2001 The behavior of mixed poly(methacrylic acid)/poly(ethylene oxide) (PMA/PEO) shells of hybrid polymeric micelles with polystyrene cores was studied in detail by a combination of light scattering, fluorometric, potentiometric, and other techniques. The results show that the dissociation of poly(methacrylic acid) in the inner layer close to the polystyrene core is suppressed, in part because of the relatively low polarity of the medium and because of a high concentration of carboxylic groups. The dissociation degree does not correspond to the bulk pH. The PMA chains form a hydrogen-bond-stabilized interpolymer complex with PEO chains in the inner shell. The compact layer of the PMA-PEO complex around the polystyrene core is very stable and resistant to changes in the bulk solvent properties. Potentiometric titration shows that an important fraction of PMA, which is engaged in the complex formation, cannot be neutralized even in a considerable excess of the base. The peripheral part of the layer is formed preferentially by the stretched and ionized free ends of the PMA blocks. Hybrid PS-(PMA/PEO) micelles resemble so-called “onion type” micelles, for example, polystyrene-block-poly(2-vinylpyridine-block-poly(ethylene oxide)) [see: Procha´zka et al. Macromolecules 1996, 29, 6526.] and may be regarded as “pseudo-multilayer” polymeric nanoparticles.

Introduction Block polyelectrolyte micelles formed by amphiphilic samples with strongly hydrophobic blocks, for example, polystyrene, PS, and polyelectrolyte blocks such as poly(methacrylic acid), PMA, behave as kinetically frozen, electrically charged nanoparticles in aqueous media.1-3 PS-PMA micelles contain small nonpolar PS cores and the shells formed of the fairly stretched and partially ionized PMA blocks.2a,e The PS cores are in the glassy state.2b The properties of the micellar solutions are controlled by the polyelectrolyte behavior of the shell. A number of experimental studies indicate that the inner part of the polyelectrolyte PMA shell close to the nonpolar core behaves differently from the peripheral part because of the specific properties of the PMA homopolymer.2a,d,3,4 * To whom correspondence should be addressed. † This study forms part of the long-time Research Plan of the School of Science of Charles University in Prague, “Structure, dynamics and function of molecular and supramolecular assemblies”, MSM 11310001. ‡ Supported by the Ministry of Education of the Czech Republic, Grant VS 97 103. (1) Tuzar, Z.; Kratochvı´l, P. In Surface and Colloid Science; Matievic, E., Ed.; Plenum Press: New York, 1993; Vol. 15, p 1. (2) (a) Kiserow, D.; Procha´zka, K.; Ramireddy, C.; Tuzar, Z.; Munk, P.; Webber, S. E. Macromolecules 1992, 25, 461. (b) Tian, M.; Quin, A.; Ramireddy, C.; Webber, S. E.; Munk, P.; Tuzar, Z.; Procha´zka, K. Langmuir 1993, 9, 1741. (c) Sˇ teˇpa´nek, M.; Podha´jecka´, K.; Procha´zka, K.; Teng, Y.; Webber, S. E. Langmuir 1999, 15, 4185. (d) Sˇ teˇpa´nek, M.; Procha´zka, K. Langmuir 1999, 15, 8800. (e) Sˇ teˇpa´nek, M.; Procha´zka, K.; Brown, W. Langmuir 2000, 16, 2502. (f) Munk, P.; Procha´zka, K.; Tuzar, Z.; Webber, S. E. CHEMTECH 1998, 20. (3) (a) Sˇ teˇpa´nek, M.; Krijtova´, K.; Procha´zka, K.; Teng Y.; Webber, S. E.; Munk, P. Acta Polym. 1998, 49, 96. (b) Sˇ teˇpa´nek, M.; Podha´jecka´, K.; Tesarˇova´, E.; Procha´zka, K.; Tuzar, Z.; Brown, W. Langmuir 2001, 17, 4240.

Efforts aimed at broadening the range of potential applications require design, preparation, and study of novel types of “tailor-made” water-soluble micelles with tunable properties. The preparation of hybrid polymeric micelles is one of several new strategies in applied polymer colloid research. The functional properties of hybrid micelles depend on composition and may be easily tuned depending on the intended use. Experimental difficulties with the preparation of hybrid micelles arise mainly from the fact that the overwhelming majority of polymers differing in chemical nature are incompatible but the preparation of stable hybrid micelles requires their spontaneous formation. It is therefore necessary to identify suitable water-soluble polymers that are sufficiently compatible. Two widely used polymers, PMA and poly(ethylene oxide), PEO, provide a promising pair.5 In this paper, we describe the behavior of mixed watersoluble shells of hybrid polymeric micelles. Their preparation was described in the preceding paper.3b The study was performed by a combination of light scattering, fluorometry, NMR, and potentiometric titration. Despite the fact that studies of water-soluble polymeric micelles (4) (a) Katchalski, A. J. Polym. Sci. 1951, 7, 393. (b) Arnold, R. J. Colloid Sci. 1957, 1, 549. (c) Anufrieva, E. V.; Birshtein, T. M.; Nekrasova, T. N.; Ptitsyn, C. B.; Scheveleva, T. V. J. Polym. Sci., Part C 1968, 16, 3519. (d) Delben, F.; Crezcenzi, V.; Quadrifoglio, F. Eur. Polym. J. 1972, 8, 933. (e) Koenig, J. L.; Angood, A. C.; Semen, J.; Lando, J. B. J. Am. Chem. Soc. 1969, 91, 7250. (f) Wang, Y.; Morawetz, H. Macromolecules 1986, 19, 1925. (g) Bedna´rˇ, B.; Trneˇna´, J.; Svoboda, P.; Vajda, Sˇ .; Fidler, V.; Procha´zka, K. Macromolecules 1991, 24, 2054. (5) (a) Zeghal, M.; Auvray, L. Europhys. Lett. 1999, 45, 482. (b) Mathur, A. M.; Drescher, B.; Scranton, A. B.; Klier, J. Nature 1998, 392, 367. (c) Iliopoulos, I.; Audebert, R. Eur. Polym. J. 1998, 24, 171. (d) Bekiranov, S.; Bruinsma, R.; Pincus, P. Europhys. Lett. 1993, 24, 171.

10.1021/la010247p CCC: $20.00 © 2001 American Chemical Society Published on Web 06/06/2001

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are common in the past decade,6 the preparation and detailed studies of systems with hybrid micellar cores or shells are very rare.7 Experimental Section (a) Materials. Block Copolymer Samples. The block copolymer samples used and their characterization were given in the preceding paper.3b Block Polyelectrolyte Micelles. The preparation of both singlecomponent and hybrid micelles with mixed PMA/PEO shells was described in detail in the preceding paper.3b DAF. 5-(N-Dodecanoyl)aminofluorescein, DAF, was purchased from Molecular Probes and used as obtained. (b) Experimental Techniques. Quasielastic and Static Light Scattering. The ALV setup, which was used for both static and quasielastic light scattering (QELS) measurement, preparation of solutions for measurement, details of the measurement, and the data evaluation was described in the preceding communication.3b Apparent hydrodynamic radii of the micelles, RHap, were measured in the angular range between 30° and 150° at low, albeit finite, concentrations, below 0.1 mg/mL, that is, in the concentration region where intermicellar interactions may be neglected. UV-Vis Absorption Measurements. Measurements of the UVvis absorption spectra were performed in 1 cm quartz cuvettes using a Hewlett-Packard HPUV 8452A diode array spectrophotometer. NMR Measurements. 1H NMR spectra were registered using the Varian apparatus UNITYINOVA 400 (proton frequency 400 MHz). Spectra of polyelectrolyte micelles were measured in aqueous buffers with addition of 5% of D2O. Either 1,4-dioxane (3.53 ppm) or 2-methyl-2-propanol (1.25 ppm) was used as the internal standard. Presaturation of the H2O signal was used for 0.5 s before the measurement. The block copolymer sample, PSPEO, was dissolved in CDCl3, and tetramethylsilane was used as the internal standard. Steady-State Fluorometry. Steady-state fluorescence spectra (i.e., corrected excitation and emission spectra and steady-state anisotropy) were recorded with a SPEX Fluorolog 3 fluorometer in a 1 cm quartz cuvette closed with a Teflon stopper. Oxygen was removed by 5 min of bubbling with nitrogen before the measurement. Time-Resolved Fluorometry. The time-correlated single photon counting technique was used for measurements of fluorescence lifetimes. The time-resolved fluorescence decays were recorded on an Edinburgh Instruments ED 299 T time-resolved fluorometer, equipped with a nanosecond coaxial discharge lamp filled with hydrogen at 0.5 atm (half-width of the pulse ca. 1.2 ns).3a The apparatus allows for a multiplexing regime of the simul(6) (a) Wilhelm, M.; Zhao, C.-L.; Wang, Y.; Xu, R.; Winnik, M. A.; Mura, J.-L.; Riess, G.; Croucher, M. D. Macromolecules 1991, 24, 1033. (b) Rager, T.; Meyer, W. H.; Wegner, G.; Winnik, M. A. Macromolecules 1997, 30, 4911. (c) Zhao, C.-L.; Winnik, M. A.; Riess, G.; Croucher, M. D. Langmuir 1990, 6, 514. (d) Astafieva, I.; Zhong, X. F.; Eisenberg, A. Macromolecules 1993, 26, 7339. (e) Astafieva, I.; Khougaz, K.; Eisenberg, A. Macromolecules 1995, 28, 7127. (f) Yu, Y. S.; Zhang, L. F.; Eisenberg, A. Langmuir 1997, 13, 2578. (g) Zhang, L. F.; Eisenberg, A. Macromolecules 1999, 32, 2239. (h) Shen, H. W.; Zhang, L. F.; Eisenberg, A. J. Am. Chem. Soc. 1999, 121, 2728. (i) Shen, W. H.; Eisenberg, A. Macromolecules 2000, 33, 2561. (j) Antonietti, M.; Heinz, S.; Schmidt, M.; Rosenauer, C. Macromolecules 1994, 27, 3276. (k) Antonietti, M.; Fo¨rster, S.; Hartmann, J.; O ¨ strich, S. Macromolecules 1996, 29, 3800. (l) Antonietti, M.; Fo¨rster, S.; O ¨ strich, S. Macromol. Symp. 1997, 121, 75. (m) Antonietti, M.; Go¨ltner, C. Angew. Chem., Int. Ed. Engl. 1997, 36, 910. (n) Regenbrecht, M.; Akari, S.; Fo¨rster, S.; Mohwald, H. J. Phys. Chem. B 1999, 103, 6669. (p) Buthun, V.; Lowe, A. B.; Billingham, N. C.; Armes, S. P. J. Am. Chem. Soc. 1999, 121, 4288. (r) Lee, A. S.; Gast, A. P.; Buthun, V.; Armes, S. P. Macromolecules 1999, 32, 4302. (s) Huang, H. Y.; Kowalewski, T.; Remsen, E. E.; Gertzmann, R.; Wooley, K. L. J. Am. Chem. Soc. 1997, 119, 11653. (t) Wooley, K. L. J. Polym. Sci. 2000, 38, 1397. (u) Wu, G.; Zhou, Z.; Chu, B. Macromolecules 1993, 26, 2117. (7) (a) Erhardt, R.; Bo¨ker, A.; Abetz, V.; Mu¨ller, A.; Stadler, R. Janus Micelles. Proceedings of the World Polymer Congress IUPAC Macro 2000, Warsaw, July 9-14, 2000; p 470. (b) Since the first submission of the manuscript, the following paper on “Janus micelles” appeared: Erhardt, R.; Bo¨ker, A.; Zettl, H.; Kaya, H.; Pychhout-Hintzen, W.; Kraush, G.; Abetz, V.; Mu¨ller, A. H. E. Macromolecules 2001, 34, 1069.

Figure 1. Dependencies of the apparent hydrodynamic radius, RHap, on the molar fraction of PMA units in shells, xPMA, for hybrid PS-(PMA/PEO) micelles and mixtures of pure PSPMA and PS-PEO micelles (curves 1 and 2 for the hybrid micelles and curves 3 and 4 for the mixtures). Measurements were made in alkaline buffers, pH 9.2, differing in ionic strength: I ) 0.015 mol/L (1,3) and I ) 0.68 mol/L (2,4). Copolymer concentration, cp ) 0.1 mg/mL. taneous acquisition of fluorescence and excitation profiles (SAFE). A reconvolution procedure was used to get the true fluorescence decays that were further fitted to multiexponential functions using the Marquardt-Levenberg nonlinear least-squares method. Low values of the χ2 (close to 1.0) and random distributions of residuals were used as criteria of the fit. Potentiometric Measurements. The pH measurements were performed using a PHM 93 reference pH meter, Radiometer, Denmark, equipped with a combined glass microelectrode PHC 2406. Carbonate-free solutions of NaOH were used, and all measurements and manipulations were performed in a N2 atmosphere.

Results and Discussion Light Scattering Measurements. The polyelectrolyte behavior of the shell of hybrid and parent micelles was studied by QELS. Representative QELS results are depicted in Figure 1. Curve 1 shows apparent hydrodynamic radii, RHap, measured at low, albeit finite, concentrations of hybrid micelles in a low-ionic-strength alkaline buffer (I ) 0.015 mol/L, pH 9.2) as a function of the molar fraction of PMA units in the shell, xPMA. The RHap value for the PS-PEO micelles is fairly small, RHap ) 20 nm, whereas that for PS-PMA micelles is considerably larger, RHap ) 56 nm. This observation is consistent with results of techniques used in the preceding paper.3b The value for PS-PMA micelles corresponds well to that measured earlier in the same buffer.2e The RHap values for the hybrid micelles increase steeply with increasing content of PMA and level off for xPMA higher than 0.4. Curve 2 shows the RHap versus xPMA curve for hybrid micelles in a relatively high ionic strength alkaline buffer (I ) 0.68 mol/L, pH 9.2). The experimental value for xPMA ) 0, that is, for nonelectrolyte PEO micelles, does not depend on the ionic strength of the solution, whereas that for xPMA ) 1, that is, for PS-PMA micelles, is strongly depressed by ionic strength. Curves 3 and 4 show the average values, RHap, for mixtures of individual micelles under conditions corresponding to curves 1 and 2, respectively. The appreciably smaller PS-PEO micelles scatter light more weakly than the larger PS-PMA micelles and contribute only little to the measured intensity. Because the measured radius, (RHap)-1, is the z-average of reciprocal radii of individual micelles, the curve for the mixture rises faster in the region of low xPMA values than that for hybrid micelles. The analysis of the distribution of relaxation

Hybrid Polymeric Micelles in Aqueous Media

Figure 2. Dependencies of (RHap)I/(RHap)I)0.015 on the ionic strength, I, of pure PS-PEO micelles (curve 1), hybrid PS(PMA/PEO) micelles with xPMA ) 0.05 and 0.5 (curves 2a and 3a, respectively), PS-PMA and PS-PEO mixtures with xPMA ) 0.05 and 0.5 (curves 2b and 3b, respectively), and pure PSPMA micelles (curve 4). Measurements were made in an alkaline buffer, pH 9.2; copolymer concentration, cp ) 0.09 mg/mL.

times, τA(τ), for hybrid micelles always led to one narrow peak indicating the presence of uniform particles in the solutions. Values of the second moment of the peak, Γ, were proportional to sin2(θ/2) which proves the diffusive character of the relaxation mode. The thickness of the polyelectrolyte shell depends on pH and on the ionic strength of the solution. We have shown in our earlier studies that the long-range interactions between micelles (mostly the counterion mediated effects) may be ruled out at the low micellar concentration used (that is, ca. 102 times lower than that of the copolymer).2c-e,3b The shell expansion is controlled mostly by the entropy-to-enthalpy balance of counterions that have to sacrifice a part of their translation freedom in order to compensate the shell charge.8 It is interesting to compare the collapse of the shell induced by small ions for pure PMA and the mixed PMA/PEO shell. Figure 2 shows the dependencies of the relative apparent hydrodynamic radius on the ionic strength of the alkaline buffer (pH 9.2), for PS-PEO micelles (curve 1), PS-PMA micelles (curve 4), and hybrid PS-(PMA/PEO) micelles with xPMA ) 0.05 and 0.5 (curves 2a and 3a, respectively). Plotted values are normalized by pertinent maximum values, measured for the corresponding micelles in very dilute buffers (I ) 0.015 mol/L). The curve for pure PS-PMA micelles compare well with our data published earlier.2e Experimental data show that changes in size become less pronounced with increasing content of PEO in the shell. Curves 2b and 3b show analogous dependencies for mixtures of individual micelles with xPMA ) 0.05 and 0.5, respectively. Fluorometric Measurements. Important information on the shell behavior may be obtained by steady-state and time-resolved fluorescence measurements, using fluorescent surfactants that bind in the inner shell close to the core/shell interface, for example, DAF.2c,d In this work, we use DAF for probing the mixed polyelectrolyte/ nonelectrolyte shell. All fluorometric measurements were (8) (a) Pincus, P. Macromolecules 1991, 24, 2912. (b) Israels, R.; Leermakers, F. A. M.; Fleer, G. J.; Zhulina, E. B. Macromolecules 1994, 27, 3249. (b) Borisov, O. V.; Zhulina, E. B.; Birsthein, T. M. Macromolecules 1994, 27, 4795. (c) Shusharina, N. P.; Nyrkova, I. A.; Khokhlov, A. R. Macromolecules 1996, 29, 3167. (d) Shusharina, N. P.; Linse, P.; Khokhlov, A. R. Macromolecules 2000, 33, 3829. (e) Misra, S.; Mattice, W. L.; Napper, D. H. Macromolecules 1994, 27, 7090. (f) Groenewegen, W.; Ugelhaaf, S. U.; Lapp, A.; van der Maarel, J. R. C. Macromolecules 2000, 33, 3283. (g) Karymov, M. A.; Procha´zka, K.; Mendelhall, J.; Martin, T. J.; Munk, P.; Webber, S. E. Langmuir 1996, 12, 4748.

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Figure 3. Dependencies of the DAF emission maximum wavelength, λmax, on the probe-to-micelle molar ratio, ξ, for alkaline solutions (pH 9.2, I ) 0.05 mol/L). Curve 1 corresponds to pure PS-PMA micelles, curve 2 corresponds to hybrid PS(PMA/PEO) micelles with xPMA ) 0.73, and curve 3 corresponds to hybrid micelles with xPMA ) 0.5. Concentration of DAF, c ) 1.5 µmol/L. Inset: Maximum fluorescence intensities, IF, as functions of ξ for the same systems as in Figure 3.

performed in an alkaline borate buffer (pH 9.2, I ) 0.15 mol/L) because both types of probes, that is, the micellebound and the water-dissolved, are fluorescent at this high pH. The emission maximum wavelengths, λmax, of DAF versus ξ are plotted in Figure 3, for hybrid micelles with different shell compositions and a constant fluorophore concentration, cF ) 1.5 µmol/L. Corresponding fluorescence intensities, IF versus ξ, are shown in the inset. Curves 1, 2, and 3 correspond to pure PS-PMA and to hybrid micelles with xPMA ) 0.73 and 0.50, respectively. Amphiphilic DAF shows very high affinity for PS-PMA, PS-PEO, and hybrid micelles. At low probe-to-micelle ratios, ξ, the microphase equilibrium may be described by a constant apparent partition coefficient, Kpap, which is quite high (Kpap ) ca. 105 for PS-PMA micelles).2d At polymer concentrations of ca. 1 mg/mL and low ξ of ca. 101, almost all probes bind to the micelles. They occupy the most favorable positions with their hydrocarbon tails immersed partially in the core and partially in the hydrophobic inner layer of the shell and the fluorescent headgroups in the transition region between the hydrophobic and hydrophilic parts of the shell. With increasing ξ, the shell-sorbed DAF molecules spread through the whole shell. At high ξ, the shell saturates and a considerable amount of DAF molecules dissolve in the aqueous phase. The shell-sorbed probes are close to each other, and their fluorescence is self-quenched because of the formation of H-aggregates. These aggregates are not only nonfluorescent, but they play the role of very efficient traps in the process of excitation energy migration.2d,9 The water-dissolved DAF molecules are exposed to a polar aqueous medium, and their fluorescence and absorption are thus blue-shifted.2c,d For pure PS-PMA micelles, the center of the sigmoidal part of the λmax curve and the minimum in the IF curve are attained at ξ ) ca. 200. At low probe-to-micelle ratios, the fluorescence maximum at 522 nm indicates that the probes experience a fairly nonpolar microenvironment in the inner shell. The maximum position remains constant up to ξ ) ca. 100; then, it starts to drop and at ξ ) ca. 500 (9) (a) McRae, E. G.; Kasha, M. J. Chem. Phys. 1958, 28, 721. (b) Kasha, M.; Rawls, H.; El-Bayoumi, M. Pure Appl. Chem. 1965, 11, 37. (c) Dutta, K.; Salesse, C. Langmuir 1997, 13, 5401.

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Figure 4. Time-resolved fluorescence decays for DAF in alkaline solutions of PS-(PMA/PEO) micelles with xPMA ) 0.5 for different ξ. Curve 1 correspond to a low ξ ) 2, and curve 2 corresponds to a high ξ ) 810; curve 3 shows the excitation profile. Concentration of the polymer, cP ) 19 mg/L (curve 1) and cP ) 0.05 mg/L (curve 2); concentration of the probe, cDAF ) 1.5 µmol/L; pH 9.2; I ) 0.15 mol/L. Inset: The mean fluorescence lifetime, τF, as a function of ξ.

it attains a value of 513 nm, which is typical for fluorescein emission in alkaline buffers. Fluorescence intensity decreases between ξ ) 10 and 200 because of the formation of H-aggregates (resulting, in part, from a decreasing concentration of the fluorescent species and from efficient dynamic quenching of fluorescence from nonaggregated DAF molecules by nonradiative excitation energy transfer to H-aggregates). The quenching is quite pronounced, and the fluorescence intensity is reduced ca. 4 times at ξ ) ca. 200. With a further increase in ξ, the emission intensity rises as a result of the contribution from the waterdissolved probes. λmax remains constant over the whole ξ-region where the emission intensity drops considerably. It means that the concentration of water-dissolved probes is very low and their contribution to the emission is negligible up to almost full saturation of the shell by ca. 200 DAF molecules. For mixed systems, all λmax curves start at 519 nm. They resemble those for pure PS-PMA micelles, but their sigmoidal parts shift to lower ξ with decreasing xPMA. For high probe-to-micelle ratios, ca. 500, the drop levels off and attains a constant value, λmax ) 513 nm, for all (mixed as well as single-component) micellar systems, indicating that the active fluorescent probes experience an aqueous microenvironment. As concerns the IF curves, their minima become considerably shallow and the corresponding wavelength shifts to lower ξ for micelles with low xPMA. It suggests that the sorption capacity of mixed shells for DAF molecules is small as compared with that of the pure PMA shell and decreases with decreasing xPMA. Representative results of time-resolved fluorescence measurements with DAF sorbed to hybrid micelles with xPMA ) 0.5 are depicted in Figure 4. Curve 1 shows the fluorescence decay for a system with a low DAF-to-micelle ratio, ξ ) 2. The decay is essentially single-exponential, and the mean fluorescence lifetime, τF ) ca. 4.6 ns, corresponds to a relatively “hydrophobic” and dense microenvironment in the inner shell. Curve 2 shows the decay for a system with ξ ) 810. In this case, most probes are dissolved in the aqueous phase. Their decay is almost single-exponential, and the mean fluorescence lifetime, τF ) ca. 4.0 ns, is only slightly shorter than that for the shell-sorbed probes. Curve 3 shows the excitation profile. The dependence of τF on ξ is shown in the inset. The curve

Podha´ jecka´ et al.

Figure 5. The Stern-Volmer plots for the iodide quenching of the micelle-bound DAF fluorescence in an alkaline borate buffer, pH 9.2. Symbols IF0 and IF stand for emission intensities without and with the iodide, respectively, and cI- is the iodide concentration. Triplets of curves 1, 2, and 3 for PS-PMA micelles and curves 4, 5, and 6 for hybrid PS-(PMA/PEO) micelles with xPMA ) 0.5 depict quenching in solutions with ξ ) 20, 150, and 300, respectively.

is constant for ξ ) 1-10, and then it drops in the region between 10 and 50 and remains constant for higher ξ. In contrast to the analogous curve for pure PS-PMA micelles, it does not pass through a minimum.2c,d This difference in fluorescence behavior is due to the small sorption capacity of hybrid micelles as compared with PS-PMA micelles. With increasing ξ, the partition equilibrium shifts rapidly in favor of the water-dissolved probes and the contribution of the fluorescence from the shell-sorbed DAF molecules is no longer important (cf. the shallow minimum in curve 3 in the previous figure). Additional information may be obtained by the quenching of the DAF fluorescence by iodide anions.10 The SternVolmer plots, that is, the dependencies of (IF0/IF) on cI(where IF0 and IF are the fluorescence intensities measured without and with the quencher, respectively, and cI- is the iodide concentration), are shown in Figure 5. Curves 1 and 2 correspond to pure PS-PMA micelles and small and medium ξ values, 20 and 150, respectively. For ξ ) 20, the probes are embedded in a compact part of the shell. They are inaccessible to iodide anions, and the quenching efficiency is negligible. In the system with ξ ) 150, the fluorescence is strongly self-quenched (i.e., the IF0 is small) and the additional iodide quenching is also negligible. The Stern-Volmer plot for a system with a considerable excess of DAF with respect to PS-PMA (curve 3, ξ ) 300) shows efficient quenching. In this case, emission from the shell-sorbed probes is self-quenched, irrespective of iodide presence or absence. Emission from the waterdissolved DAF is efficiently quenched by the water-soluble quencher. Curves 4, 5, and 6 depict the fluorescence quenching in hybrid PS-(PMA/PEO) micelles (xPMA ) 0.5) for ξ ) 20, 150, and 300, respectively. If we compare the curves for PS-PMA and PS-(PMA/PEO), it is evident that quenching is easier in mixed micellar systems. The difference between PS-PMA and mixed PS-(PMA/PEO) (10) The iodide anion is a negatively charged quencher, and its penetration in the negatively charged PMA shell is hindered. The quenching efficiency for the shell-bound probes is therefore lower than that in the solution, but we have found in our earlier studies that it is sufficient for the purpose. It disturbs the shell less than the cationic quencher and monitors not only the structure but also the charge of the shell. It would have been advantageous to compare quenching caused by anionic and cationic quenchers. However, fluorescein dyes are very little quenched by cations because cations interact preferentially with the COOH group and not with the fluorescent part of the molecule.

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Nevertheless, the sigmoidal increasing part does not correspond to full titration of PMA in the system. It means that the innermost part of the PMA shell is never neutralized, even in strongly alkaline solutions with a considerable surplus of the base. When comparing parts a and b of Figure 6, it is necessary to keep in mind that both micellar solutions contain the same molar concentration of PMA despite the fact that they differ in the composition of the shell. The comparison shows clearly that the fraction of PMA that may be neutralized decreases with increasing content of PEO in the shell. This finding is consistent with results of other techniques that show that the interpolymer PMA/PEO complex in the inner part of the shell is very stable and resistant to changes in the bulk conditions. Concluding Remarks

Figure 6. Time-dependent titration curves for an aqueous solution of (a) PS-PMA micelles and (b) hybrid PS-(PMA/ PEO) micelles with xPMA ) 0.5. Molar concentration of the poly(methacrylic acid), cPMA ) 0.85 g/L; concentration of the added NaOH, cNaOH ) 0.1 mol/L. Measurements were made at different times after NaOH addition: 20 min (curve 1), 2 h (curve 2), 1 day (curve 3), 4 days (curve 4), 11 days (curve 5), and 22 days (curve 6).

systems is due to a lower sorption capacity of the mixed shell and a consequent shift of the DAF partition equilibrium in favor of the water-dissolved probes which are easily accessible to the water-soluble iodide ions. Time-Dependent Alkalimetric Titration of Micellar Shells. Alkalimetric titration provides a fundamental characterization of the micellar shells. Because the density of the inner shell is fairly high, the pH-induced conformation changes proceed very slowly. In an earlier paper,2e we measured the time-dependent titration curves of PSPMA micelles over a period of several weeks. In the present work, we have extended the time-dependent titration to mixed systems. Results of titration are depicted in parts a and b of Figure 6 for PS-PMA and hybrid PS-(PMA/ PEO) micelles with xPMA ) 0.5, respectively. Concentrations of micelles were adjusted in order to keep the molar concentration of PMA constant, which means that the equivalent amounts of the base are the same in all cases. After the addition of NaOH, only the water-exposed carboxylic groups at the shell periphery are neutralized instantaneously. The COOH groups in the inner shell are hidden in the hydrophobic layer, and only a small fraction of the hydroxide is consumed immediately after its addition. The nonconsumed NaOH causes an important pH increase. At longer times, the alkaline cations slowly disturb the collapsed inner layer of the shell and further COOH groups are exposed and neutralized. The consumption of base results in a considerable pH drop. The changes proceed on the time scale of days which is quite surprising for the shell thickness of only ca. 10-20 nm. Within one month, the titration curve stops changing.

For a correct understanding of the mixed shell behavior, it is necessary to keep in mind several facts: (i) PEO blocks in the studied system are appreciably shorter than PMA blocks. (ii) The interpolymer PMA/PEO complex is formed under conditions where the PMA is protonated. (iii) The polarity of the inner shell is low because of the proximity of the nonpolar PS core, and dissociation of carboxyl groups in a narrow layer around the core is suppressed even in highly basic buffers (with pH higher than 10). The results of the present study together with the above-mentioned facts suggest the following model for the mixed shell: (a) The PMA-Rich Mixed Shell. The inner layer is formed by the interpolymer PMA/PEO complex, whereas the shell periphery is formed preferentially by PMA. Carboxylic groups in the inner layer do not dissociate, and the hydrogen atoms are engaged in the complex with PEO. Peripheral COOH groups are exposed to the polar aqueous medium, and their dissociation corresponds roughly to the bulk pH. In low-ionic-strength alkaline solutions, the degree of dissociation of COOH is almost independent of xPMA. In mixed systems, the PMA periphery of the shell is more dilute as compared with pure PSPMA micelles (see Scheme 1 in ref 3b) which promotes COOH dissociation. This model may explain the maximum in electrophoretic mobility for micelles with xPMA ) ca. 0.5 that was observed in CZE measurements.3b (b) The PEO-Rich Mixed Shell. A considerable fraction of PMA is engaged in the complex with PEO. The fraction of COOH groups, which may be potentially ionized, is relatively low and the degree of dissociation, as well as the electrophoretic mobility, decreases with decreasing xPMA. The complex is very stable, and the COOH groups engaged in the complex are protected against neutralization. At low pH, almost all PEO is firmly bound in the complex and the shell structure is fairly dense and rigid. At high pH, relative short peripheral segments of both PEO and PMA chains do not form the complex, which may be demonstrated by NMR spectra. Curve a in Figure 7 shows the 1H NMR spectrum of the molecularly dissolved block copolymer PS-PEO in CDCl3. The narrow peak at ca. 3.65 ppm corresponds to protons in CH2 next to oxygen in the PEO block. Curves b, c, and d depict expanded parts of the spectra for hybrid micelles (xPMA ) 0.5) in aqueous buffers at pH 9.2 and pH 5.3 and pure PS-PEO micelles in an aqueous buffer at pH 5.3, respectively. The peak for protons in CH2O groups is shifted to lower values, ca. 3.40 ppm, because of changes of the microenvironment dielectric properties. The main difference between the spectra of the molecularly dissolved and the micellized PEO is a considerable broadening of the CH2O peak because of very limited mobility of polymer

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the latter case, almost all PEO forms the complex with PMA and the mobility of PEO segments in the compact interpolymer complex is very low. All data suggest that the shell may be regarded as a “pseudo-two-layer” system. The properties of the inner and outer shells differ considerably. The transition region appears to be fairly narrow.11 The state of the shell periphery is controlled by the polyelectrolyte behavior of PMA. The inner part is formed by the fairly compact and stable neutral PMA/PEO complex. Its behavior is almost independent of bulk pH and ionic strength. The thicknesses of the inner and the outer layers depend on the PMA-to-PEO weight ratio and on the bulk aqueous buffer.

Figure 7. 1H NMR spectra of (a) PS-PEO solution in CDCl3, PS-(PMA/PEO) hybrid micelles (xPMA ) 0.5) in an aqueous buffer (b) at pH 5.3 and (c) at pH 9.2, and (d) pure PS-PEO micelles in an aqueous buffer at pH 5.3.

segments in the shell in all micellar systems. In pure PSPEO micelles, there is still an important fraction of relatively mobile segments and the peak is not too broad. In the mixed shell, the peak is broad in alkaline solutions, but it disappears fully into the noise level at low pH. In

Acknowledgment. This study was supported by Charles University Grant 215/2000/BCH/PrˇF. M. Sˇ teˇpa´nek would like to acknowledge support from the Swedish Institute during his study stay at Uppsala University. The authors thank Associate Professor Dr. Jan Sejbal from the NMR Laboratory of Charles University in Prague for NMR spectra measurements. LA010247P (11) The very low sorption capacity of mixed shells for DAF shows that the thickness of the layer in which DAF may be sorbed is strongly limited as compared with pure PS-PMA micelles.