Time-Dependent Behavior of Block Polyelectrolyte Micelles in

Block polyelectrolyte samples containing long hydrophobic blocks, e.g., polystyrene, and long ... Supported by Ministry of Education of the Czech Repu...
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Langmuir 2000, 16, 2502-2507

Time-Dependent Behavior of Block Polyelectrolyte Micelles in Aqueous Media Studied by Potentiometric Titrations, QELS and Fluorometry 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, 128 40 Prague 2, Czech Republic

Wyn Brown Department of Physical Chemistry, Uppsala University, Box 532, 751 21 Uppsala, Sweden Received July 29, 1999. In Final Form: November 9, 1999 Block polyelectrolyte samples containing long hydrophobic blocks, e.g., polystyrene, and long polyelectrolyte blocks, e.g., poly(methacrylic acid), do not dissolve in aqueous media. However, multimolecular micelles consisting of compact polystyrene cores and polyelectrolyte shells may be prepared by dialysis from organic solvent-water mixtures into aqueous buffers. Polystyrene cores are kinetically frozen in water and the behavior of the micellar systems is determined by the polyelectrolyte behavior of the shell. Poly(methacrylic acid) does not represent a typical polyelectrolyte. Due to the presence of a strongly hydrophobic methyl group in each repeating unit, its behavior more resembles that of a polysoap. In aqueous micellar systems, poly(methacrylic acid) chains form a fairly compact hydrophobic layer close to the core/shell interface. In our recent works, we have found that the state of the inner part of the shell responds rather slowly to shock changes in the bulk pH and the ionic strength of the solution. In this work, we use a combination of potentiometry, QELS, and fluorometry to study the time-dependent behavior of polyelectrolyte micellar shells.

Introduction Block copolymers containing a long, strongly hydrophobic block, such as polystyrene, PS, and a long polyelectrolyte block, such as poly(methacrylic acid), PMA, do not dissolve in aqueous buffers. However, it is possible to prepare multimolecular micelles consisting of compact PS cores and PMA shells in aqueous media indirectly. Copolymer samples may be dissolved, e.g., in a 1,4-dioxanewater mixture rich in 1,4-dioxane, which is a selective solvent for polystyrene and the micelles may be transferred into water-rich solvents by stepwise dialysis.1 Since water is a very strong precipitant for PS, micellar cores are in a nonequilibrium kinetically frozen state in aqueous media2 and properties of aqueous micellar systems depend strongly on the polyelectrolyte behavior of the PMA shell.3 In our laboratory, we have been studying the micellization of block copolymers in selective solvents both experimentally2-6 and theoretically7 for a fairly long time. In our * To whom correspondence should be addressed. † Supported by Ministry of Education of the Czech Republic (Grant No. VS 97 103). (1) Tuzar, Z.; Webber, S. E.; Ramireddy, C.; Munk. P. Polym. Prepr., Am. Chem. Soc., Div. Polym. Chem. 1991, 32, 525. (2) Tian, M.; Quin, A.; Ramireddy, C.; Webber, S. E.; Munk, P.; Tuzar, Z.; Procha´zka, K. Langmuir 1993, 9, 1741. (3) Kiserow, D.; Procha´zka, K.; Ramireddy, C.; Tuzar, Z.; Munk, P.; Webber, S. E. Macromolecules 1992, 25, 461. (4) (a) Tuzar, Z.; Kratochvı´l, P.; Procha´zka, K.; Contractor, K. Makromol. Chem. 1989, 190, 2967. (b) Procha´zka, K.; Vajda, Sˇ .; Fidler, V.; Bedna´rˇ, B.; Mukhtar, E.; Almgren, M.; Holmes, S. J. Mol. Struct. 1990, 219, 377. (c) Procha´zka, K.; Manda´k, T.; Kocˇirˇ´ık, M.; Bedna´rˇ, B.; Tuzar, Z. J. Chem. Soc, Faraday Trans. 1990, 86, 1103. (d) Procha´zka, K.; Manda´k, T.; Bedna´rˇ, B.; Trneˇna´, J.; Tuzar, Z. J. Liquid Chromatogr. 1990, 13, 1765. (e) Tuzar, Z.; Konˇa´k, C ˇ .; Sˇ teˇpa´nek, P.; Plesˇtil, J.; Kratochvı´l, J.; Procha´zka, K. Polymer 1990, 31, 2118. (f) Procha´zka, K.; Bedna´rˇ, B.; Mukhtar, E.; Svoboda, P.; Trneˇna´, J. Almgren, M. J. Phys. Chem. 1991, 95, 4563.

recent papers,2,5,6 we have mainly used static and quasielastic light scattering, SANS, and both steady-state and time-resolved fluorometry for studying high-molarmass block polyelectrolytes in aqueous media. We have found that the dissociation of the carboxylic groups in the micellar shell is suppressed and does not correspond to the bulk pH of the solvent. Properties of the shell vary continuously in the radial direction from the core/shell interface toward the micellar periphery. The dissociation of -COOH groups increases and the density of the shell segments decreases. The innermost part of the shell is very compact and relatively hydrophobic even at neutral and slightly alkaline pH6e and resembles the hypercoiled hydrophobic domains formed by linear PMA at low pH.8 (5) (a) Procha´zka, K.; Kiserow, D.; Ramireddy, C.; Tuzar, Z.; Munk, P.; Webber, S. E. Macromolecules 1992, 25, 454. (b) Ramireddy, C.; Tuzar, Z.; Procha´zka, K.; Webber, S. E.; Munk, P. Macromolecules 1992, 25, 2541. (c) Procha´zka, K.; Martin, T. J.; Munk, P.; Webber, S. E. Macromolecules 1996, 29, 6518. (d) Karymov, M. A.; Procha´zka, K.; Mendenhall, J. M.; Martin, T. J.; Munk, P.; Webber, S. E. Langmuir 1996, 12, 4749. (e) Plesˇtil, J.; Krˇ´ızˇ, J.; Procha´zka, K.; Webber, S. E.; Munk, P.; Wignall, G. Macromolecules, accepted. (6) (a) Sˇ teˇpa´nek, M.; Krijtova´, K.; Procha´zka, K.; Teng, Y.; Webber, S. E., Munk, P. Acta Polymer. 1998, 49, 96. (b) Sˇ teˇpa´nek, M.; Krijtova´, K.; Limpouchova´, Z.; Procha´zka, K.; Teng, Y.; Munk, P.; Webber, S. E. Acta Polym. 1998, 49, 103. (c) Teng, Y.; Morrison, M.; Munk, P.; Webber S. E.; Procha´zka, K. Macromolecules 1998, 31, 3578. (d) Krijtova´, K.; Sˇ teˇpa´nek, M.; Procha´zka, K.; Webber, S. E. J. Fluoresc. 1998, 8, 21. (e) Sˇ teˇpa´nek, M.; Krijtova´, K.; Procha´zka, K.; Teng, Y.; Webber, S. E. Colloids Surfaces A: Physicochem. Eng. Aspects 1999, 147, 79. (f) Sˇ teˇpa´nek, M.; Podha´jecka´, K.; Procha´zka, K.; Teng, Y.; Webber, S. E. Langmuir 1999, 15, 4185. (g) Sˇ teˇpa´nek, M.; Procha´zka, K. Langmuir 1999, 15, 8800. (7) (a) Limpouchova´, Z.; Procha´zka, K. Collect. Czech. Chem. Commun. 1993, 58, 2290; 1994, 59, 803. (b) Procha´zka, K.; Limpouchova´, Z. Collect. Czech. Chem. Commun. 1994, 59, 782; 1994, 59, 2166. (c) Procha´zka, K. J. Phys. Chem. 1995, 99, 14108. (d) Viduna, D.; Limpouchova´, Z.; Procha´zka, K. Macromolecules 1997, 30, 7263. (e) Limpouchova´, Z.; Viduna, D.; Procha´zka, K. Macromolecules 1997, 30, 8027.

10.1021/la9910226 CCC: $19.00 © 2000 American Chemical Society Published on Web 01/29/2000

Time-Dependent Behavior of Block Polyelectrolyte Micelles

Various nonpolar compounds may be solubilized not only in the core, but also in the inner part of the shell.6 The amount of the nonpolar compound solubilized in the shell is sometimes comparable with that solubilized in PS cores. The release of the core solubilized molecules is usually very slow. The shell solubilized molecules may be released considerably faster than those solubilized in cores. In recent years, polymeric micelles have also been studied as agents for the controlled uptake and release of biomedicinally, or environmentally important compounds.9 A proper understanding of the uptake and release kinetics requires a knowledge of the time-dependent relaxation processes in micellar solutions. Due to a fairly high segment density of PMA in the inner part of the shell, all conformational changes of chains in the hydrophobic layer caused by changes of the bulk pH, ionic strength of the solution, etc., proceed rather slowly.8k In this paper, we study the slow response of the shell to sudden changes of the bulk pH by a combination of potentiometric titration, QELS, and fluorometry. Experimental Section Copolymer Samples. Polystyrene-block-poly(methacrylic acid) diblock copolymer sample, PS-PMA, weight-average molar mass, Mw ) 4.4 × 104 g/mol, Mw/Mn ) 1.05, weight fraction of polystyrene, wPS ) 0.68, was synthesized by Dr. T. J. Martin. Details of the sample preparation and characterization are given in ref 5b. Polymeric PS-PMA micelles were prepared by stepwise dialysis from 1,4-dioxane/water mixtures into the alkaline borate buffer (pH 9.3). The preparation procedure is given elsewhere.6a To remove the traces of the alkaline buffer as much as possible, micellar solutions were dialyzed against 0.05 M NaCl for one month, before titrations and other measurements. Polystyrene-block-poly(ethylene oxide) diblock copolymer, PSPEO, weight-average molar mass, Mw ) 2.1 × 104 g/mol, weight fraction of polystyrene, wPS ) 0.46, was synthesized by Dr. C. Ramireddy. Polymeric PS-PEO micelles were prepared by stepwise dialysis from 1,4-dioxane/water mixtures into water. 5-(N-dodecanoyl)aminofluorescein, DAF, Molecular Probes, USA, was used as obtained. Steady-State Fluorometry. Steady-state fluorescence spectra were recorded on a SPEX Fluorolog 3 fluorometer. Potentiometric Titrations. The pH titrations were performed using a PHM 93 reference pH meter, Radiometer, Denmark, and a combined PHC 2406 glass microelectrode. Quasielastic Light Scattering. The light scattering setup consists, as described previously,10a of a 488 nm Ar ion laser light source and the detector optics coupled via a monomodal fiber to an ITT FW 130 photomultiplier. The ALV-PM-PD amplifierdiscriminator was connected to an ALV-5000 autocorrelator/ computer. The cylindrical scattering cells were sealed after filtration through 0.22 µm Millipore filters and immersed in a (8) (a) Katchalski, A. J. Polymer. 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) Ghiggino, K. P.; Tan, K. L. In Polymer Photophysics; Phillips, D., Ed.; Chapman and Hall: London, 1985; Chapter 7. (g) Tan, K. L.; Treolar, F. E. Chem. Phys. Lett. 1980, 73, 239. (h) Bedna´rˇ, B.; Morawetz, H.; Shafer, J. A. Macromolecules 1985, 18, 1940. (i) Wang, Y.; Morawetz, H. Macromolecules 1986, 19, 1925. (j) Bedna´rˇ, B.; Trneˇ`ına´, J.; Svoboda, P.; Vajda, Sˇ .; Fidler, V.; Procha´zka, K. Macromolecules 1991, 24, 2054. (k) Schorr, P.; Toomey, R.; Tirrell, M.; Cook, D.; Mays, J. W. in Polyelectrolytes; Noda, I., Kokufuta, E., Eds.; Yamada Science Foundation: Osaka, Japan, 1999. (9) (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. (d) Nagarajan, R.; Barry, M.; Ruckenstein, E. Langmuir 1986, 2, 210. (e) Nagarajan, R.; Ganesh, C. Macromolecules 1989, 22, 4312. (f) Tonkisakis, A.; Hilfiker, R.; Chu, B. J. Colloid Interface Sci. 1982, 90, 1033. (10) (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.

Langmuir, Vol. 16, No. 6, 2000 2503 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 relation10b

g2(t) - 1 ) β|g1(t)|2

(1)

where β is a factor accounting for deviation from the 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)dlnτ

(2)

where t is the lag time. The relaxation time distribution, τA(τ), is obtained by performing the inverse Laplace transform with the aid of a constrained regularization algorithm (REPES),10c 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 ) (4πno/λ)sinθ/2 is the magnitude of the scattering vector and Γ ) 1/τ is the relaxation rate. Here θ is the scattering angle, n0 the refractive index of pure solvent and λ the wavelength of the incident light. Within the dilute regime, D varies linearly with the polymer concentration (C), that is,

D ) D0 (1 + kD C)

(3)

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

D0 ) kBT/6πηoRH

(4)

where kBT is the thermal energy factor and η0 is the temperaturedependent viscosity of the solvent.

Results and Discussion The polyelectrolyte shell of the multimolecular PSPMA micelle represents a very specific polyelectrolyte assembly in aqueous media that does not behave as in a normal polyelectrolyte solution. The shell-forming PMA blocks are anchored to the kinetically frozen core and may be regarded as a convex polyelectrolyte brush. A number of fairly successful theories of polyelectrolyte brushes have been published recently.11 However, linear PMA does not represent a typical polyelectrolyte. The conformational behavior of PMA was studied by a number of research groups and it was shown that polymer chains undergo strong hypercoiling with decreasing pH in the region of pH 6 to 5.8 Polyelectrolyte behavior of micellar shells formed by PMS blocks is very complex. Indirect fluorometric data suggest that the inner layer of the shell is considerably more dense and hydrophobic than the hypercoiled microdomains in linear PMA. This hydrophobic layer in the inner shell persists up to a fairly high (11) (a) Misra, S.; Mattice, W. L. Macromolecules 1994, 27, 2058. (b) Mattice, W. L. In Polymer and Solvent Organization; Webber, S. E., Munk, P., Tuzar, Z., Eds.; NATO ASI: Dordrecht, The Netherlands, 1995. (c) Seidel, C. Macromolecules 1994, 27, 7085. (d) Israe¨ls, R.; Leermakers, F. A. M.; Fleer, G. L. Macromolecules 1994, 27, 3087. (e) Lyatskaya, Y. V.; Leermakers, F. A. M.; Fleer, G. J.; Zhulina, E. B.; Birshtein, T. M. Macromolecules 1995, 28, 3562. (f) Zhulina, E. In Polymer and Solvent Organization; Webber, S. E., Munk, P., Tuzar, Z., Eds.; NATO ASI: Dordrecht, The Netherlands, 1995.

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Figure 1. pH of PS-PMA samples (sample volume 0.5 mL, cP ) 1.4 g/L) as a function of the volume of added 0.2 M NaOH, VNaOH, measured immediately (curve 1) and at 2 days (curve 2), 7 days (curve 3), 25 days (curve 4), and 68 days (curve 5) after the addition of the base.

bulk pH. Due to the Donnan equilibria,12 the apparent acidity of the shell PMA is reduced as compared with the linear PMA, and the transition from the collapsed to the stretched regime appears at relatively high pH. This transition concerns mainly the middle part of the chain and may be observed by QELS, in measurements of the hydrodynamic radius of PS-PMA micelles, RH, as a function of pH.5c Potentiometric Titration of PS-PMA Micelles. Alkalimetric titration of polyelectrolyte micelles allows a fundamental characterization of the micellar shell. Due to the high density of the inner part of the shell, the pHinduced conformational changes proceed rather slowly. This is the reason we have measured the time-dependent titration curves over several weeks. The time-dependent pH measurement was performed in the following way: The aqueous solution of PS-PMA micelles was divided into several 0.5 mL portions. Increasing amounts of alkaline hydroxide were added to each solution and the pH was measured as a function of time after the base addition. The maximum molar amount of the added base equals twice the equivalent amount of the polyacid. The carbonate-free solution of the base was used and all measurements and manipulations were performed in a N2 atmosphere to prevent the formation of carbonates in the solution. The conventional titration curves of polyelectrolyte micelles, i.e., pH as a function of the added base, measured at different times after addition of the base are shown in Figure 1 for solutions with a low ionic strength (I ) 0.05, NaCl). Curve 1 was obtained immediately after the base addition. Other curves were measured later. The time-dependent changes in the curve shapes are fairly pronounced and may be observed over a time range of days and weeks. They may be explained as follows. In the PMA shell, the degree of -COOH dissociation decreases, while the segment density and the microenvironment hydrophobicity increase from the shell periphery toward the core/shell interface. The water-exposed carboxylic groups at the periphery of the shell are neutralized instantaneously. Carboxylic groups in the dense inner part of the shell are hidden in hydrophobic domains and are not affected at short times. It means that only a relatively small fraction of the hydroxide is consumed in neutralization immediately after addition of the base. The nonconsumed OH- causes a fast pH increase. At later times, the alkaline cations slowly disturb the collapsed inner layer of the shell which results in the (12) Munk, P.; Tuzar, Z.; Procha´zka, K. Collect. Czech. Chem. Commun. 1997, 62, 1730.

S ˇ teˇ pa´ nek et al.

Figure 2. pH of PS-PMA samples (sample volume 0.5 mL, cP ) 1.4 g/L), as a function of time after addition of 5 µL (curve 1), 10 µL (curve 2), 15 µL (curve 3), 20 µL (curve 4), and 50 µL (curve 5) of 0.2 M NaOH.

exposure of further -COOH groups. The consequent neutralization consumes an equivalent amount of OHanions and the bulk pH drops. The most pronounced timedependent pH-changes are observed at the beginning of titration, i.e., with small additions of the base. One aspect requires a comment. The starting pH of micellar solutions is fairly high (almost pH 6). This high value is due, in major part, to the above-mentioned Donnan equilibria that affect the acidity of the shell, and, in minor part, to the preparation procedure. Micelles are prepared by stepwise dialysis from 1,4-dioxane/water mixtures into an aqueous buffer. Dialysis into slightly alkaline buffers results in a lower polydispersity of the prepared aqueous solutions of PS-PMA micelles than that performed directly into water. The final aqueous solutions were dialyzed for one month against the 0.05 M NaCl prior to pH measurements. As a consequence of the slow relaxation processes in the shell, it is almost impossible to exchange small traces of counterions from the previous buffer solution and to obtain the theoretical value of pH. However, if we take into account the apparent value of pKa in the PMA shell ca. 7, and the actual PS-PMA concentration ca. 1.4 g/L, the pH difference (corresponding to the difference between the theoretical and measured concentrations of H3O+ ions ca. 10-5 mol/L) does not represent a significant discrepancy. Figure 2 shows five time dependencies of the bulk pH for micellar solutions differing in amounts of the added base (5 to 50 µL of the 0.2 M NaOH). Addition of the base results in a fast increase of pH that is followed by a slow decrease to the equilibrium value. For a correct discussion of the time-dependent changes of PMA chains in the micellar shell, it is necessary to remember that curves in Figure 2 show pH, i.e., the logarithm of the H3O+ activity vs time, and that they have to be recalculated into the true consumption of the added base to obtain an understanding of the shell behavior. Curve 1 (for 5 µL of the added 0.2 M NaOH to 0.5 mL of 1.4 g/L PS-PMA) shows an instantaneous jump to pH 9 and a relatively fast decrease by almost 2 pH units that levels off within 2-3 days. The decreasing part of the curve corresponds to a fairly fast breakdown of relatively loose hydrophobic domains and neutralization of -COOH groups in the middle shell. The pH drop is fairly pronounced, but corresponds to a decrease in the concentration of the bulk OH- by only 10-5 mol/L. Curve 2 (10 µL of NaOH) shows a jump to pH 11.2 and a slow decrease to pH 7.8 which corresponds to the consumption of OH- giving a change in concentration in the bulk solution by 1.6 × 10-3 mol/L. The pH decrease is caused by a slow disruption of a considerable part of the inner PMA layer and by neutralization of a high fraction of the exposed carboxylic

Time-Dependent Behavior of Block Polyelectrolyte Micelles

groups. It proceeds very slowly (over weeks) due to the fact that the inner part of the shell is very compact and hydrophobic. The hydrophobicity of this layer is affected by the proximity of the nonpolar PS core. The effective dielectric permittivity of the core/shell interface and of the innermost PMA layer is fairly low. Addition of 15 and 20 µL of NaOH (curves 3 and 4, respectively) results in a jump to a pH of about 11.6 and a slow decrease to a pH of ca. 8.3 and 9.5, respectively. In solutions with a high surplus of the base, the changes caused by time-dependent neutralization of the continuously exposed -COOH groups are small as compared with the total concentration of the base. This is the reason an addition of 50 µL of NaOH (curve 5), results in an almost completely instantaneous pH changesno slow decrease in pH is observed. The initial jumps are not shown in the figure since the inclusion of vertical lines would lead to less comprehensible graphs. The time dependent pH curves indicate that the disruption of the middle part of the shell is facile and proceeds quite rapidly in comparison with the disruption of the inner shell, but it is still slow as compared with the diffusion-controlled processes in structures with nm dimensions. The measurements performed suggest that a shock addition of a considerable surplus of the base causes rather fast but incomplete conformational changes in the shell (which are followed by slower changes), while a small amount of the added base results in less pronounced and much slower changes. Nevertheless the full dissociation of the shell PMA is impossible even with a high surplus of base since the thin hydrophobic layer next to the core is strongly affected by the low polarity of the core, and is fairly resistant to the pH-induced perturbation. A very peculiar cascade-like breakdown of the shell has been observed during alkalimetric titration of the PSPMA micelles in solutions containing Li+ ions.13 This behavior is fairly reproducible, but supplementary detailed studies are needed to identify the reasons for this behavior. Swelling of the shell may be affected, e.g., by the preferential sorption of various small counterions, by the length of the PMA block, by the preparation procedure, etc. Quasielastic Light Scattering Study. The pHdependent conformational changes and dissociation of the carboxylic groups in the shell result in a partial stretching of the shell-forming blocks and in a measurable increase in the micellar size.5c QELS represents one of the most reliable standard techniques for studying the polymeric micellar systems and we believe that the time-dependent change in the size of micelles measured by QELS provides the most convincing proof of the slow pH-induced behavior of the shell. Representative results of the time-dependent QELS measurement are shown in Figure 3 (curve 1). The curve corresponds to the addition of a small amount of base which causes a fast but rather limited stretching of the middle part of the shell. It is evident that the increase in RH is well-measurable and the curve is smooth. Curve 2 shows the corresponding pH change. Comparison of curves 1 and 2 shows that the RH increase is coupled with neutralization of PMA as expected on the basis of the above outlined model of the shell. However two aspects are slightly surprising: (i) The addition of base causes a sudden decrease in the micellar size (not shown) followed by a slower increase. (ii) The RH change proceeds considerably faster than the pH change. This apparent paradox may be understood on the basis of theoretical (13) Procha´zka, K.; Sˇ teˇpa´nek, M.; Webber, S. E. In Polyelectrolytes; Noda, I., Kokufuta, E., Eds.; Yamada Science Foundation: Osaka, Japan, 1999.

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Figure 3. Hydrodynamic radius, RH (curve 1) and pH (curve 2), of PS-PMA sample (sample volume 0.5 mL, cP ) 1.4 g/L), as a function of time after the addition of 5 µL of 0.2 M NaOH.

density profiles of polyelectrolyte brushes for different values of the ionic strength.11 Calculations by Zhulina and Fleer based on the mean-field theory predict that the density of the inner part of the weak polyelectrolyte brush is high at low ionic strength, but the free ends of chains are dissociated and fairly stretched. With increasing ionic strength, the periphery part of the brush first collapses and then at a higher ionic strength, the middle part of the shell starts to swell. The shock addition of the base triggers a process that proceeds with time via the above-described intermediate stages. The sudden decrease in RH corresponds to the collapse of the micelle periphery. The relatively fast recovery and the further increase in RH is again connected with conformational changes at the micelle periphery, while the slower pH decrease requires conformational changes and dissociation of carboxylic groups in a relatively thick layer of the shell. Spectroscopic Studies with the Interface-Bound DAF. Steady-state and time-resolved fluorescence studies were performed to obtain detailed information on the inner part of the PMA shell. A pH-sensitive amphiphilic probe, 5-(N-dodecanoyl)aminofluorescein (DAF), which has a strong affinity for nonpolar surfaces, was used. It binds to micelles with its aliphatic chain buried in the core and the fluorescein headgroup localized in the core-shell interfacial region. Here we are interested in the timedependent behavior of micelles. In our earlier papers, we have investigated UV-Vis absorption and fluorescence properties of fluorescein-based fluorescent surfactants, 5-(N-dodecanoyl)aminofluorescein, DAF, and 5-(N-octadecanoyl)aminofluorescein, OAF, in detail. We have studied behavior of the probes in aqueous media,6f their binding to polymeric micelles (the binding equilibrium6d,e,g and binding kinetics6e) and fluorescence properties of the PS-PMA micelle-bound probes6g very carefully. The fluorescence properties of both DAF and OAF are basically similar to those of fluorescein, however nonnegligible differences at concentrations higher that 10-6 M for OAF and 10-4 M for DAF are due to fairly low solubility in aqueous media and to the surfactant nature of probes (easy formation of nonfluorescent H-aggregates and strong self-quenching of fluorescence). They are weak acids (containing groups COOH and OH) and belong to the category of current fluorescent pH indicators. Fluorescent species are double-dissociated dianions, while all other forms are nonfluorescent. It means that considerable changes in fluorescence intensity with pH are due to changes in the dissociation equilibrium and to changing concentration of dianions, while the fluorescence lifetime almost does not change with pH. In our earlier studies, we have measured fluorescence lifetimes of DAF and OAF over a wide range of experi-

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mental conditions.6f,g Actual lifetimes are little affected by binding to polymeric micelles, e.g., the values measured for DAF are: (τF)M ) 4.6 ns for the PS-PMA micellebound DAF and (τF)W ) 4.3 ns for DAF in an alkaline borate buffer, pH ca. 9. The measured values almost do not depend on pH (values range from 4.3 to 4.5 ns which is almost in the range of experimental error). As mentioned above, both DAF and OAF may form H-dimers or higher aggregates either in aqueous solutions (quite easily for OAF), or in micellar shells (when sorbed). These aggregates are not only nonfluorescent, but act as very efficient excitation-energy traps.6g This means that at high fluorophore loading of micelles, a dynamic fluorescence quenching, manifested both by a decrease in fluorescence intensity and a related decrease in fluorescence lifetime, may be observed. In this study we confine to low loading (ca. 10 DAF molecules per micelle) and we do not have to take this effect into consideration. Experimental curves for the micelle-bound DAF were measured after a full equilibration of the micellar solutions. The full equilibration of the system assumes (i) an accomplished equilibrium binding of the probe to micelles and (ii) an accomplished swelling of micellar shells with respect to the bulk pH (which may be accompanied by a subsequent probe redistribution between micelles and the bulk solvent and by its partial redistribution within the shell). (i) To achieve the equilibrium binding, we add a small volume (typically 10 µL) of 10-4 M solution of DAF in an alkaline borate buffer, pH 9.3, to 1 mL of the micellar solution in the same buffer, I ca. 0.1) and leave the mixture to equilibrate overnight. In our older studies, we have found that the binding kinetics of OAF to micelles is fairly fast (i.e., the binding is accomplished within few minutes) in solutions with low ionic strength, while it proceeds on the time scale of hours in salt-containing solutions.6e This slightly surprising finding is a result of a fairly complex behavior of OAF in aqueous solutions. It may be understood with the help of our detailed studies of the probe behavior in aqueous media.6g The probe has very low solubility in aqueous media due to the presence of a long hydrophobic tail and it forms dimers and lower associates at very low concentrations (ca. 10-6 M) depending on the ionic strength. The driving force for the association arises from hydrophobic interactions, while electrostatic repulsion between ionized and negatively charged fluorescent headgroups hinders this process. The presence of small ions in the solutions screens electrostatic interactions and promotes association. In the binding process, the OAF monomeric molecules penetrate into micellar shells and bind in the interfacial core/shell region. In media with a high ionic strength, concentration of the free OAF monomer is low and the binding of the fluorophore to micelles competes with the association equilibrium in the bulk solvent. This is why the binding process is hindered and slowed. As concerns DAF, the binding kinetics is similar but faster and differences in the binding rate with ionic strength are significantly less pronounced since the nonpolar tails are shorter and the probe is more soluble in water and in aqueous buffers than OAF. With respect to the above given facts, overnight mixing should allow for a full equilibrium binding of DAF to micelles. (ii) As concerns the time-dependent response of the shell to pH changes, we have found that it is very slow when a base is added (see the preceding section); however a process of a moderately slow stepwise dialysis from alkaline solutions in the direction of decreasing pH (ca. 0.5 pH unit per 2 h) yields micellar solutions that do not further change. In this work, we have measured only a few supplementary curves necessary for understanding the fluo-

S ˇ teˇ pa´ nek et al.

Figure 4. Fluorescence titration curves of free DAF (curve 1), DAF/PS-PMA system (curve 2) and DAF/PS-PEO system (curve 3). Concentrations of DAF and polymer were cDAF ) 1.5 µM and cP ) 0.7 g/L, respectively.

rescence behavior. The normalized fluorescence intensity (excit. at 490 nm, emiss. at 520 nm) as a function of pH for the free (curve 1) and the PS-PMA (curve 2), and PS-PEO micelle-sorbed DAF (curve 3) are shown in Figure 4 for systems containing small numbers of probes per micelle (ca. 10 DAF molecules). The decrease in the apparent acidity of the -OH group after the DAF binding to the core/shell interface is manifested by a considerable shift of the sigmoidal part of the curve toward higher pH as compared with the free and the PS-PEO micelle-bound DAF. This shift is consistent with the Fromherz theory.14 The difference between the pKa of the DAF bound to the anionic PS-PMA and the neutral PS-PEO micelle allows calculation of the interfacial potential in the anionic micelles. In contrast to systems studied by Fromherz et al. that contain probes sorbed on the surfactant micelles, there is no distinct electric double layer at the surface of the polymeric micelles. Dissociation varies smoothly within the shell and DAF molecules monitor the properties of the innermost part of the shell. Nevertheless, the apparent conventional electrostatic potential, φ(ap), may be evaluated at the core/shell interface. The measured electrostatic potential has to be interpreted with a great caresit is necessary to keep in mind that the shell-forming PMA is a weak polyelectrolyte and the average dissociation of -COOH in shells varies with pH. The true electrostatic potential at the core/shell interface depends therefore on pH. A fairly complex behavior of the probe at the interface is manifested also by a lower dependence of fluorescence intensity on pH than obtained for free DAF (the electrostatic effect of the pH-independent surface potential should result in a shift of the sigmoidal part of the curve, which should not change the slope). The apparent value of φ(ap) ) -51 mV was determined from the inflection points of the curves for PS-PEO and PS-PMA micelles. This value seems to be a very reasonable estimate for polymeric micelles15 and explains the fairly high electrophoretic mobilities of polymeric micelles with PMA shells.16 The fluorescence emission and the fluorescence anisotropy of the micelle-bound DAF monitor the timedependent conformational changes in the inner part of the PMA shell. However, they also provide information on the binding of DAF to the micelles. The dependencies of the fluorescence emission of the PS-PMA bound DAF on time after the addition of the base are shown in Figure (14) Ferna´ndez, M. S.; Fromherz, P. J. Phys. Chem. 1977, 81, 1755. (15) Almost the same values were obtained by electrophoretic light scattering, ζ-potential was found to be ca. -50 mV (Procha´zka, K.; Webber, S. E.; Munk, P. unpublished data). (16) Tuzar, Z.; Procha´zka, K.; Zuskova´, I.; Munk, P. Polym. Prepr. 1993, 34, 1038.

Time-Dependent Behavior of Block Polyelectrolyte Micelles

Figure 5. Fluorescence intensity, IF (curve 1), and fluorescence anisotropy, 〈r〉 (curve 2), of the DAF/PS-PMA system (sample volume 3 mL, cP ) 1.4 g/L, cDAF ) 3 µM), as a function of time after addition of 30 µL 0.2 M NaOH.

5. Changes in the measured fluorescence intensity, IF (curve 1), and anisotropy, 〈r〉 (curve 2), proceed on the same time scale as the pH changes, although both the sudden initial jumps and the slow changes at longer time seem to contradict theoretical expectation. The steadystate fluorescence anisotropy, 〈r〉, is fairly high before addition of the base since the inner part of the shell is very compact. It drops rapidly upon addition of NaOH, and it increases slowly at longer times. The initial drop from the value 〈r〉 ) 0.26, before addition, to the value 〈r〉 ) 0.15 immediately after the addition of the base, is not shown in order not to obscure the curves. This behavior may be explained as follows. The headgroup of the DAF is a large fluorescein ring. The fluorescently active form is a double-ionized fluorescein headgroup and therefore we assume that it is located just in the transition region between the hydrophobic and the moderately hydrophilic part of the shell. Ionization phenomena and steric hindrance compete with the sorption driving force, i.e., with favorable interactions of the nonpolar aliphatic chain of DAF with the PS core. The equilibrium distance of the charged headgroup from the core/shell interface is the result of this complex competition. Similar distributions of amphiphilic fluorophores that depend on the interplay between the probe affinity to different microphases and electrostatic phenomena occur in various biological structures and are used in biochemistry, e.g., in membrane studies with charge-sensitive probes.17 Immediately upon addition of NaOH, the middle part of the shell breaks down and the free parts of the shell-forming blocks become more extended. The polarity and the local pH of the probe microenvironment increases and the microviscosity decreases, which causes a sudden drop in fluorescence anisotropy and a rise in fluorescence intensity. At later times, the inner hydrophobic part of the shell slowly breaks down. It allows the DAF headgroup to penetrate closer to the core. This process is manifested by a slow increase in the fluorescence anisotropy and decrease in the fluorescence intensity. Fluorescence anisotropy reflects the rotational mobility of the whole fluorescein ring and (17) Pla´sˇek, J.; Sigler, K. J. Photochem. Photobiol. B: Biology 1996, 33, 101, and references therein.

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Figure 6. Comparison between pH dependence of normalized fluorescence intensity, IF/IFmax, and fluorescence anisotropy, 〈r〉, of DAF/PS-PMA system relaxing after addition of NaOH (conditions as in Figure 5) and the same system in equilibrium at given pH (conditions as in Figure 4). Curves 1 and 2, respectively, refer to IF/IFmax and 〈r〉 of the relaxing system, and curves 3 and 4 to IF/IFmax and 〈r〉 of the equilibrated system.

therefore monitors the compactness of the larger microenvironment. The fluorescence intensity, on the other hand, is proportional to the degree of dissociation of the -OH group and monitors mainly the state of the first solvatation shell of the -OH group. This is the reason changes in fluorescence intensity proceed faster than those in anisotropy. In Figure 6, the time-dependent IF(t) and 〈r(t)〉 values are plotted vs the time-dependent pH(t)scurves 1 and 2, respectively. The analogous dependencies of IF and 〈r〉 on pH for the fully relaxed equilibrium systems are depicted in curves 3 and 4, respectively. The significant differences between the corresponding curves show that the microenvironment of the DAF headgroup is not in the equilibrium state during the time-dependent conformational change after the addition of the base. Conclusions Time-dependent potentiomentric titration of aqueous solutions of PS-PMA micelles show that the conformational changes of the shell-forming blocks and the exposure of carboxylic groups accessible for neutralization proceeds rather slowly, on the time scale of days and weeks. Three processes, differing considerably in rate, may be identified: (i) immediate neutralization of the water-exposed -COOH groups, (ii) a relatively fast breakdown of the moderately hydrophobic PMA structures in the middle part of the shell, and (iii) a slow disruption of the very hydrophobic innermost layer of the shell. All processes result in a consumption of base from the bulk solution. Changes in pH and in the micellar size may be studied by potentiometric titration and QELS measurements. Contributions of the three above-described processes to the total pH or RH changes vary with the total amount of the base added. Acknowledgment. This work was supported by Grant No. 203/97/0249 by the Grant Agency of the Czech Republic and by the Charles University Grant No. 1197/1997. LA9910226