Hybrid Polymeric Micelles with Hydrophobic Cores and Mixed

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Hybrid Polymeric Micelles with Hydrophobic Cores and Mixed Polyelectrolyte/Nonelectrolyte Shells in Aqueous Media. 1. Preparation and Basic Characterization† Miroslav Sˇ teˇpa´nek, Kla´ra Podha´jecka´, Eva Tesarˇova´, 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

Zdeneˇk Tuzar Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky´ Square 2, Prague 6 - Petrˇ iny, 168 00 Prague 6, 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 Hybrid polymeric micelles with compact polystyrene cores (PS) and mixed poly(methacrylic acid)/poly(ethylene oxide) shells (PMA/PEO) were prepared by mixing PS-PMA and PS-PEO linear diblock copolymer samples in 1,4-dioxane (80 vol %)/water. The mixture is a mild selective precipitant for PS and micelles with swollen PS cores, and either PMA or PEO shells form in both systems before mixing. Both types of micelles coexist in a mobile equilibrium with unimers and, because PMA and PEO are fairly compatible, hybrid micelles PS-(PMA/PEO) form spontaneously after mixing the individual micellar solutions. Hybrid micelles differing in the composition of the mixed shell were stepwise dialyzed into aqueous buffers. In aqueous media, the unimer-micelle equilibrium is kinetically frozen and the micelles behave as independent polymeric nanoparticles with glassy PS cores and mixed polyelectrolyte/nonelectrolyte shells. A number of experimental techniques (size-exclusion chromatography, capillary zone electrophoresis, and elastic and quasielastic light scattering measurements) were applied for characterization of the hybrid micellar systems.

Introduction Amphiphilic diblock polyelectrolytes containing a long polyelectrolyte and a long hydrophobic block do not usually dissolve in water or in aqueous buffers. However, an aqueous solution of multimolecular polymeric micelles may be prepared indirectly, for example, by stepwise dialysis from miscible mixtures of a suitable organic solvent with water into purely aqueous media.1 A typical block polyelectrolyte micelle contains a compact core formed by the insoluble nonpolar blocks and a protective shell formed by the polyelectrolyte blocks. Block polyelectrolyte micelles solubilize small hydrophobic molecules (little soluble in water) in the nonpolar cores and may be used for various environment-oriented applications, for example, for removal of nonpolar pollutants from water.2 Biocompatible water-soluble polymeric micelles (based, e.g., on block polypeptides) have been also studied for use in applications such as potential targeted-drug-delivery vehicles.3 Micellar cores formed by long hydrophobic blocks, such as high-molar-mass polystyrene, are kinetically frozen in * 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 113100001. ‡ Supported by the Ministry of Education of the Czech Republic. (1) Tuzar, Z.; Webber, S. E.; Ramireddy, C.; Munk, P. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 1991, 32, 525. (2) (a) Nagarajan, R.; Barry, M.; Ruckenstein, E. Langmuir 1986, 2, 210. (b) Nagarajan, R.; Ganesh, C. Macromolecules 1989, 22, 4312.

aqueous media. They behave as inert spheres, and the properties and stability of the micellar solutions are controlled by the polyelectrolyte behavior of the micellar shells. The polyelectrolyte micelles has been a subject of numerous studies by a number of research teams in the past decade.4 Winnik et al. have been studying watersoluble micelles by fluorometric techniques,4a,b Eisenberg et al.4c-i investigated various micellar structures formed by nonsymmetrical block polyelectrolytes by light scattering, and Fo¨rster with Antonietti4j-l studied micelles as potential nanoreactors. Billingham with Armes4m,n prepared a series of cationic and zwitterionic micelles and (3) (a) Kwon, G. S.; Naito, M.; Yokoyama, M.; Okano, T.; Sakurai, Y.; Kataoka, K. Pharm. Res. 1995, 12, 92. (b) Kataoka, K.; Kwon, G. S.; Yokoyama, M.; Okano, T.; Sakurai, Y. J. Controlled Release 1993, 24, 119. (c) Harada, A.; Kataoka, K. Macromolecules 1995, 28, 5294. (4) (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) Astafieva, I.; Zhong, X. F.; Eisenberg, A. Macromolecules 1993, 26, 7339. (d) Astafieva, I.; Khougaz, K.; Eisenberg, A. Macromolecules 1995, 28, 7127. (e) Yu, Y. S.; Zhang, L. F.; Eisenberg, A. Langmuir 1997, 13, 2578. (f) Zhang, L. F.; Eisenberg, A. Macromolecules 1999, 32, 2239. (g) Shen, H. W.; Zhang, L. F.; Eisenberg, A. J. Am. Chem. Soc. 1999, 121, 2728. (h) Shen, H. W.; Eisenberg, A. J. Phys. Chem. B 1999, 103, 9473. (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.; O ¨ strich, S. Macromol. Symp. 1997, 121, 75. (l) Regenbrecht, M.; Akari, S.; Fo¨rster, S.; Mohwald, H. J. Phys. Chem. B 1999, 103, 6669. (m) Buthun, V.; Lowe, A. B.; Billingham, N. C.; Armes, S. P. J. Am. Chem. Soc. 1999, 121, 4288. (n) Lee, A. S.; Gast, A. P.; Buthun, V.; Armes, S. P. Macromolecules 1999, 32, 4302. (p) Wooley, K. L. J. Polym. Sci. 2000, 38, 1397.

10.1021/la010246x CCC: $20.00 © 2001 American Chemical Society Published on Web 06/06/2001

Hybrid Polymeric Micelles in Aqueous Media

investigated the possibility to cross-link the polyelectrolyte shells. The cross-linking of anionic shells was studied independently by Wooley.4p Intramolecular versus intermolecular association (both closed and open) of random hydrophobically modified polyelectrolytes has been studied by a number of other teams, for example, by Morishima et al.5a,b and McCormick et al.5c,d Even if we do not touch the micellization of nonionic water-soluble polymers (mainly Pluronics) and the associative behavior of so-called “thickeners”, the number of papers on micellization of polyelectrolytes is so vast that it is futile to give all relevant references. We have been studying micellar systems based on weak block polyelectrolytes by different experimental techniques for almost 10 years, mainly in cooperation with Webber et al.6 One of several systems we have focused on was polystyrene-block-poly(methacrylic acid) micelles, PS-PMA. Poly(methacrylic acid) contains one strongly hydrophobic methyl group in each repeating unit and thus does not behave as a typical polyelectrolyte.7 “Hydrophobic” properties of PMA are very pronounced in the inner shell, in part because of low dielectric permittivity close to the PS core and also because of a considerably high concentration of monomer units. Both factors suppress the COOH dissociation, and the little-ionized PMA forms a very dense “hydrophobic” layer in the inner part of the shell around the PS core.6f,g In this paper, we describe a novel system of hybrid water-soluble micelles having PS cores and a mixed shell formed by PMA and poly(ethylene oxide), PEO, blocks. Both PMA and PEO are soluble in aqueous media over broad ranges of pH and temperature. They are compatible and form interpolymer complexes at low pH when PMA is only little dissociated.8 Formation of mixed shells and the pH-dependent complexation of PMA with PEO modify the stability and behavior of micellar systems and offer new potential applications. To our knowledge, no data on mixed polyelectrolyte micelles have been published so far, despite the fact that similar systems are very interesting from both theoretical and practical points of view.9,10 The paper describes the first part of this topic, the preparation and characterization of mixed micelles. A detailed study of the polyelectrolyte behavior of hybrid micelles with (5) (a) Szczubialka, K.; Ishikawa, K.; Morishima, Y. Langmuir 2000, 16, 2083. (b) Noda, T.; Hashidzume, A.; Morishima, Y. Macromolecules 2001, 34, 1308. (c) Smith, G. L.; McCormick, C. L. Macromolecules 2001, 34, 918. (d) McCormick et al. published a series of 78 papers on the synthesis and characterization of various water-soluble polymers and on their behavior on aqueous solutions in Macromolecules, all of them entitled “Water-soluble polymers”. (6) (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) Teng, Y.; Morrison, M.; Munk, P.; Webber, S. E.; Procha´zka, K. Macromolecules 1998, 31, 3578. (d) Sˇ teˇpa´nek, M.; Krijtova´, K.; Limpouchova´, Z.; Procha´zka, K.; Teng, Y.; Webber, S. E.; Munk, P. Acta Polym. 1998, 49, 96; 1998, 49, 103. (e) Procha´zka, K.; Martin, T. J.; Munk, P.; Webber, S. E. Macromolecules 1996, 29, 6518. (f) Sˇ teˇpa´nek, M.; Procha´zka, K. Langmuir 1999, 15, 8800. (g) Steˇpa´nek, M.; Procha´zka, K.; Brown, W. Langmuir 2000, 16, 2502. (h) Ramireddy, C.; Tuzar, Z.; Procha´zka, K.; Webber, S. E.; Munk, P. Macromolecules 1992, 25, 2541. (7) (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. (8) (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.

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mixed PMA/PEO shells will be presented in a following communication. Experimental Section (a) Materials. Copolymer Samples. Diblock copolymer samples, polystyrene-block-poly(methacrylic acid), PS-PMA, and polystyrene-block-poly(ethylene oxide), PS-PEO, were synthesized by Dr. C. Ramireddy using the living anionic copolymerization technique. Weight-average molecular weight, Mw, and PS mass fraction, xPS, of the copolymer samples were 4.2 × 104 g/mol and 0.58 for PS-PMA and 2.1 × 104 g/mol and 0.46 for PS-PEO, respectively. Details of the preparation and characterization are given in refs 4a and 6h. Preparation of Pure and Hybrid Micelles in Aqueous Solutions. Aqueous solutions of pure micelles of PS-PMA and PS-PEO were prepared by stepwise dialysis of the copolymer solutions in a 1,4-dioxane (80 vol %)/water mixture to successive mixtures having increasing contents of water. In mixtures containing less than 30 vol % dioxane, water was replaced with 0.05 M aqueous borate buffer, pH 9.2. Finally, the solutions of micelles were dialyzed several times against pure borate buffer. The same procedure was used for the preparation of hybrid micelles PS(PMA/PEO). The mixture of PS-PMA and PS-PEO copolymers dissolved in a dioxane (80 vol %)/water mixture was stirred for 48 h prior to dialysis to ensure a complete equilibration of the system. (b) Experimental Techniques. Size Exclusion Chromatography. The HPLC equipment (Watrex, Prague, Czech Republic) consisted of an DeltaChrom SDS 030 pump, a model 7125 Rheodyne injection valve (Cotati, CA) with a 20 µL sample loop, and a DeltaChrom UVD 200 variable wavelength detector. The detection wavelength was set to 214 nm. The signal acquisition and data handling were performed with the APEX integration software, version 3.0 (APEX, Prague, Czech Republic). A commercially available steel column 250 × 8 mm i.d. was used, packed with the GMB 10000 stationary phase, particle size 7 µm (Watrex, Prague, Czech Republic). The mobile phase was composed of distilled and deionized water that was degassed before use. The mobile phase flow rate was 0.5 mL/min. The measurements were carried out at a temperature of 25 °C. The samples were dialyzed into water at a concentration of 1.6 mg/mL, and 5.0 µL of these solutions was injected onto the chromatographic column. Capillary Electrophoresis. The electrophoretic measurements were carried out using a Hewlett-Packard 3D CE apparatus (Hewlett-Packard, Waldbronn, Germany), equipped with an inline variable-wavelength detector. The data were collected at three different λ-values: 214, 230, and 270 nm. A HP ChemStation (Hewlett-Packard, Waldbronn, Germany) was used to control the instrument and to process the data. An untreated silica capillary (50 µm i.d., total length 81.0 cm, length to the detector 72.5 cm) was used at an applied potential of 25 kV and a temperature of 25 °C. The background electrolyte contained 0.01 M borate buffer, pH 9.2. The capillary was conditioned sequentially with 0.1 M sodium hydroxide, with water, and finally with the buffer to be used for the subsequent separation (5 min each). The samples (dissolved in water) were injected hydrodynamically at a pressure of 10 mbar for 6 s, and the concentration was the same as in size exclusion chromatography. Thiourea was used as a marker for the electroosmotic flow. Quasielastic and Static Light Scattering. The light scattering setup consists of a 488 nm Ar ion laser light source and the detector optics coupled via a monomodal fiber to an ITT FW 130 photomultiplier as described previously.11a The ALV-PM-PD amplifier-discriminator was connected to an ALV-5000 auto(9) To our knowledge, no paper describing water-soluble micelles with mixed polyelectrolyte/nonelectrolyte shells has been published. Fairly recently, there was a poster presentation at the World Polymer Congress IUPAC Macro 2000 in Warsaw titled “Janus Micelles” by Erhardt, R., Bo¨cker, A., Abetz, W., Mu¨ller, A., and Stadler, R. The authors studied a different system with incompatible and therefore segregated shell-forming blocks. They call the micellar nanoparticles “Janus micelles” referring to a Roman god with two faces. (10) 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.

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correlator/computer. The cylindrical scattering cells were sealed after filtration through 0.22 µm Millipore filters and immersed in a large-diameter thermostated bath containing Decalin placed at the axis of a goniometer. Measurements were made at different angles, sample concentrations, and temperatures. Analysis of the data was performed by fitting the experimentally measured g2(t), the normalized intensity autocorrelation function, which is related to the electrical field correlation function, g1(t), by the Siegert relation,11b

g2(t) - 1 ) β|g1(t)|2

(1)

where β is a factor accounting for deviation from ideal correlation. For polydisperse samples, g1(t) can be written as the inverse Laplace transform (ILT) of the relaxation time distribution, τA(τ):

g1(t) )

∫τA(τ) exp(-t/τ) d ln τ

(2)

where t is the lag-time. The relaxation time distribution, τA(τ), is obtained by performing the ILT with the aid of a constrained regularization algorithm (REPES),11c which minimizes the sum of the squared differences between the experimental and calculated g2(t). The mean diffusion coefficient, D, is calculated from the second moments of the peaks as D ) Γ/q2, where q ) (4πn0/λ) sin θ/2 is the magnitude of the scattering vector and Γ ) 1/τ is the relaxation rate. Here, θ is the scattering angle, n0 is the refractive index of pure solvent, and λ is the wavelength of the incident light. Within the dilute regime, D varies linearly with the polymer concentration, C; that is,

D ) D0(1 + kDC)

(3)

where D0 is the diffusion coefficient at infinite dilution and kD is the hydrodynamic “virial” coefficient related to the solutesolute and solute-solvent interactions. The Stokes-Einstein equation relates the infinite dilution diffusion coefficient to the hydrodynamic radius, RH:

D0 ) kBT/6πη0RH

(4)

where kBT is the thermal energy factor and η0 is the temperaturedependent viscosity of the solvent. Static light scattering was performed with the same apparatus by measuring the total scattering intensity (in cps) and comparing it with that from toluene. The data were treated by the Zimm technique. Refractive index increment was calculated as the weight average of the values for corresponding homopolymers.12,13

Results and Discussion Basic Characterization of Pure and Hybrid Micellar Systems. Micellar systems were prepared by dialyzing solutions of block copolymers: (i) PS-PMA, (ii) PS-PEO, and (iii) their mixtures from 1,4-dioxane (80 vol %)/water mixtures into aqueous buffers as described in the Experimental Section. Micelles were characterized by (a) size exclusion chromatography, SEC, (b) capillary zone electrophoresis, CZE, (c) static light scattering, SLS, and (d) quasielastic light scattering, QELS. The SEC and CZE provide the most important information because it allows for differentiation of hybrid micelles from mixtures of two types of single-component micelles. We have found in our earlier studies that the PS cores of both the PSPMA and PS-PEO micelles are kinetically frozen in aqueous media and an exchange of unimer chains between micelles does not take place upon mixing the aqueous (11) (a) Schille´n, K.; Brown, W.; Johnsen, R. M. Macromolecules 1988, 27, 4825. (b) Chu, B. Laser Light Scattering, 2nd ed.; Academic Press: New York, 1991. (c) Jakesˇ, J. Czech. J. Phys. 1988, B38, 1305. (12) Milland, B.; Strazielle, C. Makromol. Chem. 1979, 180, 441. (13) Polymer Handbook; Brandup, J., Immergood, E. H., Eds.; WileyInterscience: New York, 1989.

Figure 1. SEC chromatograms of the mixture of PS-PMA and PS-PEO micelles (full curve 1) and PS-(PMA/PEO) hybrid micelles (dashed curve 2) with the PS-PMA-to-PS-PEO weight ratio of 2/1.

solutions.6b Therefore, we expect that mixing the compact multimolecular PS-PMA and PS-PEO micelles in aqueous buffers does not lead to the formation of new types of micelles with mixed PMA/PEO shells. The SEC and CZE measurements confirm our assumption. Representative results of SEC measurements are shown in Figure 1. Curve 1, the full line, shows the chromatogram for an aqueous mixture of PS-PMA and PS-PEO micelles (mass ratio 2/1) that was prepared and dialyzed separately into aqueous buffers before mixing. The PS-PMA and PS-PEO micelles differ in size and produce two wellseparated and relatively narrow peaks. Elution volumes of individual peaks correspond to those obtained in measurements with pure micelles in the same aqueous solvent. Curve 2, the dashed line, shows the chromatogram for hybrid micelles prepared by dissolution of corresponding masses of PS-PMA and PS-PEO copolymers in a mild selective solvent, 1,4-dioxane (80 vol %)/water. On the basis of our previous study6b of hybrid micelles in organic solvent/water mixtures, we assume that a fairly fast unimer-micelle exchange rate leads to the formation of hybrid micelles with PMA/PEO shells in 1,4-dioxanerich mixtures. Preliminary experiments showed that complete equilibration of the system is reached in less than 1 day. To be absolutely sure that fully equilibrated micelles have been formed, the mixtures in the mild selective mixture were stirred for 2 days. The equilibrated solution was dialyzed stepwise into the purely aqueous medium. The SEC results confirm the formation of uniform hybrid micelles. The chromatographic curve shows only one fairly narrow peak corresponding to hybrid micelles. Unfortunately, the size of hybrid micelles (see the further discussion concerning QELS data) changes only little with composition in the region of PMA-rich micelles. As a consequence, SEC cannot differentiate between the PMArich hybrid micelles and pure PS-PMA micelles because the elution volumes are almost the same. However, the obtained peak is narrow and there is no trace of a peak corresponding to pure PS-PEO micelles which provides strong evidence for the quantitative formation of hybrid micelles. To provide more direct proof of the formation of fairly monodisperse hybrid micelles over the whole composition region, capillary zone electrophoresis has been used. Almost all electrophoretic measurements were performed in a low ionic strength borate buffer (pH 9.3, I ) 0.05 mol/L). Results of measurements on a mixture of kinetically frozen PS-PMA and PS-PEO micelles (i.e., pure

Hybrid Polymeric Micelles in Aqueous Media

Langmuir, Vol. 17, No. 14, 2001 4243 Chart 1. (a) A Pure PS-PMA Micelle and (b) a Hybrid PS-(PMA/PEO) Micelle Consisting of 50% PS-PMA and 50% PS-PEO Unimers

Figure 2. CZE electrophoretic curves of (a) the mixture of PS-PMA and PS-PEO micelles and (b) PS-(PMA/PEO) hybrid micelles. The PS-PMA-to-PS-PEO weight ratio was 2/1. Inset in a: CZE electrophoretic curves of pure PS-PEO (curve 1) and PS-PMA (curve 2) micelles. Inset in b: Electrophoretic mobility, µ, of PS-(PMA/PEO) micelles, as a function of molar fraction of PMA units, xPMA, in the hybrid shells. Experimental conditions: a borate buffer, pH 9.2, ionic strength I ) 0.05 mol/L (curve 1) and I ) 0.15 mol/L (curve 2).

micelles prepared in aqueous media before mixing) are shown in Figure 2a. The electrophoretic curve shows two distinct and well-separated peaks. The first peak corresponds to nonionized PS-PEO micelles that do not migrate but move together with the basic electrolyte because of the electrokinetic flow in the quartz capillary (at the same rate as the low-molar-mass marker, thiourea).14 The second peak, moving at a considerably slower speed, corresponds to negatively charged PS-PMA micelles that, in the experimental setup used, migrate against the electrokinetic flow. The electrophoretic mobility of multimolecular PS-PMA micelles is fairly high, µ ) 3 × 10-8 m2/Vs, for their high molar mass, Mwap ) ca. 8 × 106 g/mol. Nevertheless, the effective charge on the micelles is also high; the PS-PMA micelle consists of ca. 102 of PMA chains, each containing several hundreds of carboxylic groups. A series of experimental studies show that only a fraction of peripheral carboxylic groups are ionized, depending on pH and ionic strength.6g However, even a relatively low degree of dissociation of around 10% will lead to several thousands of ionized COO- groups per micelle. The electrostatic interaction of pendant anionic groups COO- is partially screened by counterions, and the effective charge is reduced, but the remaining net charge is still high and causes an appreciably strong electrostatic force acting on the micelle in the high-voltage electrostatic field. Measurements with pure micelles under identical conditions are shown in the inset in Figure 2a. The electrophoretic mobility of pure PS-PMA micelles is the same (within experimental error) as that measured (14) Probstein, R. F. Physicochemical Hydrodynamics; John Wiley & Sons: New York, 1995.

in the mixture with PS-PEO. The mobility of pure PSPEO micelles does not change either, which suggests that intermicellar interactions are not important. Under the experimental conditions used, that is, in dilute solutions of micelles with a low ionic strength and a relatively high pH, intermicellar complexation of peripheral PMA and PEO from different micelles with formation of charged micellar clusters, (PS-PMA)n(PS-PEO)m, may be ruled out. The peripheral parts of the shell are formed mainly by PMA because the PEO entities are shorter than PMA and are engaged in complex formation in the inner shell (see below) and the ends of the PMA chains are strongly dissociated. Both factors inhibit complexation of PMA and PEO from different micelles.8 The curve shown in Figure 2b corresponds to hybrid micelles prepared by dialyzing a mixture of equal masses of PS-PMA and PS-PEO copolymers from a 1,4-dioxane (80 vol %)/water mixture into an alkaline borate buffer, pH 9.2. It shows only one peak, which is fairly narrow and corresponds to hybrid micelles. The electrophoretic mobility of the hybrid micelles is higher than that of pure PSPMA micelles under the same conditions (pH, ionic strength, temperature, concentration, etc.). This finding is slightly surprising because one intuitively expects a proportionality between the fraction of PMA in the shell and the net charge on the micelle. The mobility is a result of a competition between the net charge and the friction in the given solution. The friction depends on solvent viscosity and micellar size. Because the shell expansion depends on the dissociation of COOH groups, both the charge and friction increase with pH and the two effects partially compensate; however, the first one prevails. Light scattering measurements (see later) show that both the molar mass and the size of hybrid micelles decrease with increasing fraction of PS-PEO copolymer. The changes are small with high molar fractions of PMA in the shell and more pronounced in the region of low PMA fractions. In the system studied, the PMA chains are longer than the PEO chains and, in the mixed shell, their charged ends are stretched. The periphery of the mixed shell is formed preferentially by PMA chains, which are more dilute as compared with the pure PMA shell (see Chart 1). A lower concentration of pendant COOH groups in the periphery of the shell promotes dissociation. Hybrid micelles formed with comparable amounts of PS-PMA and PS-PEO copolymers still contain a high number of carboxylic groups per micelle. The enhanced dissociation results in an increased effective charge on the hybrid micelle and in a slightly higher electrophoretic mobility. All measurements for hybrid micelles differing in the ratio of PS-PMA to PS-PEO copolymer samples show a

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Figure 3. Molar mass, Mw (curve 1), and apparent hydrodynamic radius, RHap (curve 2), of PS-(PMA/PEO) micelles, as a function of molar fraction of PMA monomeric units, xPMA, in the shells.

single fairly narrow peak. Electrophoretic mobility as a function of the molar fraction of PMA in the shell is shown in the inset in Figure 2b. For the PMA-rich micelles in a low-ionic-strength borate buffer (pH 9.2, I ) 0.05 mol/L), it increases with decreasing amount of PMA up to the PMA molar fraction, xPMA ) 0.27. The mobility increase is not large but is well measurable, and it permits differentiation of micelles differing in PMA/PEO ratio. Electrophoretic mobility of PEO-rich micelles drops strongly with decreasing amount of PMA which suggests that even at this high pH almost all PMA is complexed with PEO and the number of dissociated COO- groups is very low considering the high micellar molar mass. The measurements show that the SEC resolution is better for the PEO-rich than for the PMA-rich micelles; however, the technique allows for a reliable proof of the formation of mixed polyelectrolyte micelles and allows investigation of their electrophoretic properties. Increasing ionic strength suppresses differences in the electrophoretic mobility in the region of the PMA-rich micelles (see curve 2). Light Scattering Measurements. Further information on hybrid micelles was obtained by static and quasielastic light scattering measurements. As mentioned above, all micellar systems studied are kinetically frozen in aqueous buffers and the association number (or molar mass) does not depend on pH, ionic strength, or temperature. However, the size of the micelles is controlled by the expansion of the polyelectrolyte shell, and it is very sensitive mainly to changes in pH and ionic strength of the buffer.15 We measured the weight-average molar mass of hybrid micelles, Mw, by static light scattering. The SLS measurements were performed in a medium-concentrated 0.05 M borate buffer in the region of polymer concentrations c ) 10-4-10-3 g/mL using an ALV apparatus. Solutions of PS-PMA, PS-PEO, and PS-(PMA/PEO) micelles are stable, and long-range interactions are suppressed in the buffer used. Results, obtained by extrapolating the experimental values to zero concentration and zero scattering angle by the conventional Zimm technique, are shown in Figure 3 (curve 1). The measured (15) (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.

values increase continuously with increasing amount of PS-PMA copolymer. The increase in Mw is pronounced in the region of low xPMA, which is consistent with results of other experimental techniques (e.g., SEC and CZE). Molar masses for pure PS-PEO and PS-PMA micelles correspond well to the values obtained earlier with a SOFICA 42 000 apparatus.6d Curve 2 shows the apparent hydrodynamic radii of micelles, RHap, measured by QELS in the borate alkaline buffer (pH 9.2, I ) 0.015 mol/L) at low, albeit finite, concentrations, ca. 1.5 × 10-4 g/mL. We have found that the RHap values are almost independent of copolymer concentration in the dilute region. Similarly to Mw, the increase in RHap is more pronounced in the region of low xPMA, and the size of the PMA-rich micelles is almost independent of the hybrid micelle composition. The shapes of the Mw and RHap versus xPMA curves are similar. For a more quantitative discussion, it is necessary to bear in mind that the length of the individual blocks and their conformational behavior differ significantly under different conditions. Because the PMA block is appreciably longer than the PEO block, an increase in the PMA content in the shell is accompanied by a relative decrease in the content of the core-forming PS with respect to the sum of the shell-forming PMA and PEO. The core is compact, and the Rcore value is therefore proportional to the third root of Mcore, which is composed of the PS blocks only. All measurements suggest that Rcore does not change much with xPMA in the studied system. The thickness of the shell and the total RHap for the PMA-rich micelles are controlled by the expansion of the PMA blocks (which are in excess in the shell) and are almost independent of the PMA/PEO ratio. As concerns the PEO-rich micelles, almost all PMA is engaged in the complex formation with PEO in the inner part of the shell and the micellar size decreases strongly with decreasing xPMA. Summary 1. Novel multimolecular polymeric micelles with PS cores and mixed (PMA/PEO) shells were prepared by mixing different ratios of PS-PMA and PS-PEO micelles in a mixture of 80 vol % of 1,4-dioxane with water. Rapid unimer exchange between individual micelles existing in this relatively mild selective solvent for both PMA and PEO allows formation of equilibrated hybrid micelles. 2. The well-equilibrated solutions were dialyzed in aqueous buffers. The polystyrene cores are kinetically frozen in aqueous media, and unimer exchange does not take place in solvents with a high water content. Individual micelles, both single-component and hybrid, are physically stabilized because of the glassy state of their cores. As a result, mixing aqueous solutions of PS-PMA and PSPEO does not produce hybrid micelles. 3. Micellar systems were characterized by SEC, CZE, and static and quasielastic light scattering. 4. Aqueous solutions of pure and hybrid micelles were studied in detail by CZE. Electrophoretic behavior of hybrid PS-(PMA/PEO) micelles was compared with that of corresponding mixtures of pure PS-PMA and PS-PEO micelles. Electrophoretic data provide direct and unambiguous proof for the formation of fairly monodisperse hybrid micelles over the whole range of PMA-to-PEO compositions. Acknowledgment. This study was supported by Charles University Grant 215/2000/BCH/PrˇF. M. Steˇpa´nek thanks the Swedish Institute for a grant supporting his stay in Uppsala. LA010246X