Fluorescence Studies on the Properties of the Nanoaggregates of Poly

Langmuir , 2003, 19 (3), pp 548–553. DOI: 10.1021/la0258852. Publication Date (Web): December 28, 2002. Copyright © 2003 American Chemical Society...
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Langmuir 2003, 19, 548-553

Fluorescence Studies on the Properties of the Nanoaggregates of Poly(ethylene oxide)-b-polymethacrylate Copolymer Formed by Binding of Cationic Surfactants to Polymethacrylate Block Yuan Li and Kenichi Nakashima* Department of Chemistry, Faculty of Science and Engineering, Saga University, 1 Honjo-machi, Saga 840-8502, Japan Received May 1, 2002. In Final Form: November 13, 2002 Fluorescence measurements have been applied to the characterization of the nanoaggregates of poly(ethylene oxide)-b-polymethacrylate (PEO-b-PMA) copolymer with the cationic surfactant cetyltrimethylammonium chloride (CTAC). It was found that the formation of the aggregates is dependent on two factors: the concentration of the block copolymer and the degree of electric charge neutralization (DN) of the carboxylate anion with cationic CTAC. The critical aggregation concentration of the complex was determined at various levels of DN. The micropolarity of the nanoaggregates was investigated by vibronic fine structure of the monomer fluorescence of pyrene (so-called I1/I3 ratio). It was revealed that the micropolarity of the aggregate particles is close to that of acetic acid or ethyl acetate. The microviscosity of the aggregates was elucidated from excimer formation of pyrene and fluorescence polarization of rhodamine B. The interior of the aggregate particle is not rigid, so that the incorporated pyrene can move around in the aggregate particle to form the excimer through a dynamic mechanism (i.e. via diffusive collision). The kinetics of the exchange of the incorporated species between the aggregate particles was examined by electronic energy transfer from rhodamine 6G to malachite green. It was found that the exchange occurs within a time scale of seconds.

Introduction Many studies have been carried out on block copolymer micelles in aqueous media in the last two decades.1-15 The block copolymer micelles have some advantages compared with conventional low molecular-weight surfactant micelles: (1) the micelle size is large, (2) various kinds of polymers can be employed both for hydrophobic and hydrophilic blocks, and (3) frozen micelles16-18 can be * To whom any correspondence should be addressed. Fax: +81952-28-8548. Phone: +81-952-28-8850. E-mail: [email protected]. (1) Ikemi, M.; Odagiri, N.; Tanaka, S.; Shinohara, I.; Chiba, A. Macromolecules 1981, 14, 34. (2) Ikemi, M.; Odagiri, N.; Tanaka, S.; Shinohara, I.; Chiba, A. Macromolecules 1982, 15, 281. (3) Zhao, C.-L.; Winnik, M. A.; Riess, G.; Croucher, M. D. Langmuir 1990, 6, 514. (4) Wilhelm, M.; Zhao, C.-L.; Wang, Y.; Xu, R. L.; Winnik, M. A.; Mura, J.-L.; Riess, G.; Croucher, M. D. Macromolecules 1991, 24, 1033. (5) Xu, R. L.; Winnik, M. A.; Hallett, F. R.; Riess, G.; Croucher, M. D. Macromolecules 1991, 24, 87. (6) Cao, T.; Munk, P.; Ramireddy, C.; Tuzar, Z.; Webber, S. E. Macromolecules 1991, 24, 6300. (7) Kiserow, D.; Prochazka, K.; Ramireddy, C.; Tuzar, Z.; Munk, P.; Webber, S. E. Macromolecules 1992, 25, 461. (8) Moffitt, M.; Khougaz, K.; Eisenberg, A. Acc. Chem. Res. 1996, 29, 95. (9) Chu, B.; Zhou, Z. Nonionic Surfactants: Polyoxyalkylene Block Copolymers; Marcel Dekker: New York, 1996; Chapter 3. (10) Almgren, M.; Brown, W.; Hvidt, S. Colloid Polym. Sci. 1995, 273, 2. (11) Alexandridis, P.; Hatton, T. A. Colloids Surf. A 1995, 96, 1. (12) Nakashima, K.; Takeuchi, K. Appl. Spectrosc. 2001, 55, 1237. (13) Nakashima, K.; Anzai, T.; Fujimoto, Y. Langmuir 1994, 10, 658. (14) Nakashima, K.; Anzai, T.; Fujimoto, Y.; Anzai, T. Photochem. and Photobiol. 1995, 61, 592. (15) Tuzar, Z.; Kratochvil, P. Surface and Colloid Science; Plenum Press: New York, 1993; Chapter 1. (16) Xu, R.; Winnik, M. A.; Riess, G.; Chu, B.; Croucher, M. D. Macromolecules 1992, 25, 644. (17) Calderara, F.; Hruska, Z.; Hurtrez, G.; Nugay, T.; Riess, G. Makromol. Chem. 1993, 194, 1411.

formed by using hydrophobic polymers with high glass transition temperature. The last property is especially advantageous when the micelles are applied to drug delivery systems.19 However, there is an important drawback in the earlier block copolymer micelles that ionic species cannot be incorporated into the core part because the core block is highly hydrophobic. Therefore, polymer micelles that can incorporate ionic species into the core have been anticipated. To overcome this problem, several research groups tried to invent new types of polymer micelles (or aggregates). One of the promising strategies is to employ a doublehydrophilic block copolymer,20-24 which consists of a polyelectrolyte in one block and a nonionic water-soluble polymer in the other. When the polyelectrolyte block is electrically neutralized with counterion, the block copolymer seems to form a micelle-like aggregate with a core of the polyelectrolyte, which can incorporate ionic species. Kataoka et al. succeeded in creating such a polymer micelle using poly(ethylene glycol)-b-poly(Llysine) and poly(ethylene glycol)-b-poly(R,β-aspartic acid) in aqueous solutions.25,26 The electrostatic interaction (18) Wang, Y.; Balaji, R.; Quirk, R. P.; Mattice, W. L. Polym. Bull. 1992, 28, 333. (19) Torchilin, V. P. J. Controlled Release 2001, 73, 137. (20) Bronstein, L.; Antonietti, M.; Valetsky, P. Nanopart. Nanostruct. 1998, 145. (21) Bronstein, L.; Sedlak, M.; Hartmann, J.; Breulmann, M.; Colfen, H.; Antonietti, M. Polym. Mater. Sci. Eng. 1997, 76, 54. (22) Bronstein, L. M.; Sidorov, S. N.; Gourkova, A. Y.; Valetsky, P. M.; Hartmann, J.; Breulmann, M.; Colfen, H.; Antonietti, M. Inorg. Chim. Acta 1998, 280, 348. (23) Bronstein, L. M.; Sidorov, S. N.; Berton, B.; Sedlak, M.; Colfen, H.; Antonietti, M. Polym. Mater. Sci. Eng. 1999, 80, 124. (24) Sidorov, S. N.; Bronstein, L. M.; Valetskt, P. M.; Hartmann, J.; Colfen, H.; Schnablegger, H.; Antonietti, M. J. Colloid Interface Sci. 1999, 212, 197. (25) Kataoka, K.; Harada, A. Science 1999, 283, 65. (26) Harada, A.; Kataoka, K. Macromolecules 1995, 28, 5294.

10.1021/la0258852 CCC: $25.00 © 2003 American Chemical Society Published on Web 12/28/2002

Nanoaggregates of PEO-b-PMA Copolymer

between the polycation and polyanion blocks provided a driving force for micelle formation. This micelle had proved to be stable and monodisperse and to be able to incorporate charged compounds (e.g., ionic drugs) into the core. However, strict size matching between poly(L-lysine) and poly(R,β-aspartic acid) blocks was needed for the formation of a stable micelle.25,26 Then, the polymer micelle (or aggregate) formation based on the interaction between double-hydrophilic block or graft copolymer and low molecular-weight ion followed, in which the size matching required in Kataoka’s case was not necessary. Kabanov et al. prepared two kinds of polymer-surfactant complexes using double-hydrophilic copolymers and ionic surfactants.27-30 One consists of the diblock copolymer of poly(ethylene oxide)-b-polymethacrylate (PEO-b-PMA) and various single-, double-, and triple-tailed cationic surfactants. The other comprises poly(ethylene oxide)-g-poly(ethyleneimine) (PEO-g-PEI) and alkyl sulfate surfactants. In the two systems, the surfactants are bound to the oppositely charged ionic segments due to the electrostatic interaction. According to Kabanov et al., the PEO-g-PEI complexes form micellelike nanoaggregates with a core from surfactant-neutralized polyions and a corona from ethylene oxide chains, while the complexes of PEO-b-PMA arrange into small vesicles having the double layers of surfactant-neutralized PMA blocks surrounded by the hydrophilic PEO blocks.27-30 Bronstein et al.20-24 reported on the formation of micelles from poly(ethylene oxide)-b-poly(ethyleneimine) (PEO-b-PEI), in which PEI block was coordinated with the transition metal compounds to form the micelle core. We also reported formation of nanoaggregates using PEO-b-PMA and alkaline earth metal ions (Ba2+ and Ca2+), in which PMA block is insolubilized by electric neutralization with the metal ions.31 Although the above-mentioned new types of polymer micelles (or aggregates) based on double-hydrophilic copolymers have attracted great interest in recent years, the details of their properties have not been elucidated. Critical micelle concentration (cmc) (or critical aggregation concentration, cac), micellar size, kinetics of exchange of unimers between micelles, and kinetics of exchange of the incorporated species between micelles (or between the micelle and bulk aqueous phase) are important properties. The dependence of such properties on outer stimuli like temperature and pH is also interesting. In this study, we employed fluorescence spectroscopy to reveal some of the properties of the nanoaggregates of PEO-b-PMA formed by binding of cetyltrimethylammonium chloride (CTAC) to PMA block. Among various aggregate parameters, we focused on the polarity of the aggregates, the mobility of the incorporated species in the aggregates, and the kinetics of exchange of the incorporated species between aggregates.

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Figure 1. Structural formulas of RB, R6G, and MG. grade), rhodamine 6G (R6G, laser grade), and malachite green (MG, guaranteed grade) from Aldrich Chemical Co. were used as supplied. Structural formulas of these three cationic dyes are shown in Figure 1. PEO-b-PMA diblock copolymer (Polymer Source Inc.) and PMA homopolymer (Scientific Polymer Products Inc.) were used as supplied. The mean degree of polymerization of PEO-b-PMA in each block is 170 for PEO and 180 for PMA. The molecular weights are Mn(PEO) ) 7500 and Mn(PMA) ) 15 500. The molecular weight of the PMA homopolymer is 150 000. Sample Preparation. Known amounts of PEO-b-PMA stock solution (with fluorescent probe, if necessary) were electrically neutralized by titration with a solution of CTAC to obtain the desired values of the degree of neutralization (DN). Here, DN is defined by

Experimental Section Materials. Water was purified with a Milli-Q purification system after it had been ion exchanged and distilled. CTAC (Tokyo Kasei) was used without further purification. Pyrene (Py, Aldrich) was purified by vacuum sublimation. Rhodamine B (RB, laser (27) Bronich, T. K.; Kabanov, A. V.; Kabanov, V. A.; Yu, K.; Eisenberg, A. Macromolecules 1997, 30, 3519. (28) Kabanov, A. V.; Bronich, T. K.; Kabanov, V. A.; Yu, K.; Eisenberg, A. J. Am. Chem. Soc. 1998, 120, 9941. (29) Bronich, T. K.; Popov, A. M.; Eisenberg, A.; Kabanov, V. A.; Kabanov, A. V. Langmuir 2000, 16, 481. (30) Bronich, T. K.; Cherry, T.; Vinogradov, S. V.; Eisenberg, A.; Kabanov, V. A.; Kabanov, A. V. Langmuir 1998, 14, 6101. (31) Li, Y.; Gong, Y.-K.; Nakashima, K.; Murata, Y. Langmuir 2002, 18, 6727.

DN (%) )

amount of added CTAC (mol) amount of COO- groups (mol)

× 100%

(1)

During titration, the samples were agitated with a magnetic stirrer to accelerate the aggregate formation. The samples were kept at room temperature for one night before the measurements. All the experiments were carried out under pH ) 6-8, where almost all carboxylic groups of PMA block have an anionic form. Turbidimetry. The turbidity was measured with a Jasco Ubest-50 UV/vis spectrophotometer. The turbidity is calculated as (100 - T)/100, where T is transmittance (%) at 350 nm. Dynamic Light Scattering Measurements. Dynamic light scattering (DLS) measurements were carried out with an Otsuka ELS-800 dynamic light scattering instrument at a fixed 90°

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Figure 2. Turbidity of PMA homopolymer and PEO-b-PMA copolymer solutions as a function of the degree of neutralization with CTAC: (O) PMA (0.02 g L-1) and (b) PEO-b-PMA (0.03 g L-1). The base molar concentration of carboxylate group is the same in PMA and PEO-b-PMA solutions. scattering angle. The sample solutions were filtered through a Millipore filter with a pore size of 0.45 µm before DLS measurements. Correlation functions were analyzed by a cumulant method and used to determine the diffusion coefficient (D) of the samples. Hydrodynamic radius (Rh) was calculated from D by using the Stokes-Einstein equation

Rh ) kBT/(6πηD)

(2)

where kB is the Boltzmann constant, T the absolute temperature, and η the solvent viscosity. Fluorescence Measurements. Fluorescence spectra were recorded on a Hitachi F-4000 spectrofluorometer. The fluorescence spectra were corrected by the use of a standard tungsten lamp with known color temperature. The excitation spectra were corrected by a conventional rhodamine B method.32 Polarization measurements were carried out with a set of polarizers.

Results and Discussion 1. Detection of the Aggregates. It is reported by Kabanov et al. that PEO-b-PMA forms nanoaggregates when the PMA block is electrically neutralized with various single-tailed cationic surfactants.28 Before we carried out fluorescence measurements, we confirmed the formation of the aggregates from PEO-b-PMA and cationic surfactant CTAC by turbidimetry and DLS measurements. Figure 2 represents the turbidity change of CTAC/PEOb-PMA copolymer system as a function of the degree of neutralization. The data for the CTAC/PMA homopolymer system are also shown for comparison. The base molar concentration of carboxylate group is the same in the two systems. In PMA homopolymer systems, the turbidity began to increase at about 40% of DN and then precipitation occurred. In contrast, the PEO-b-PMA solutions were almost transparent in the whole DN range examined. This suggests that the solubilities of the block copolymer and homopolymer complexes are quite different. The complexes of PEO-b-PMA remain soluble when the PMA block is neutralized with CTAC, because the water-soluble nonionic PEO block stabilizes the aggregates in water, whereas homopolymer PMA precipitates under the same conditions. These observations agree with those obtained in the earlier studies by Kabanov et al. on PEO-b-PMA and cationic surfactants systems.27,29 Figure 3a shows the plot of diameters (2Rh) of the aggregates as a function of the degree of neutralization with CTAC. It should be noted here that the concentration of CTAC is much lower than its cmc (1.4 mM),33 even for (32) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer Academic/Plenum Publishers: New York, 1999. (33) Kalyanasundaram, K. Photochemistry in Microheterogeneous Systems; Academic Press: New York, 1987.

Figure 3. (a) The diameter of nanoaggregate as a function of the degree of neutralization: C(PEO-b-PMA) ) 0.005 g L-1 (O), 0.01 g L-1 (b), 0.03 g L-1 (0). (b) The diameter of nanoaggregate as a function of PEO-b-PMA concentration. DN ) 100%. The counterion was CTAC for both parts a and b.

the sample with the highest DN. Therefore, CTAC does not seem to form its own micelles. From Figure 3a, the aggregate formation seems to begin at about 20% (or below 20%) of DN in the solutions with 0.01 and 0.03 g L-1 polymer concentrations. But in the solution with the concentration of 0.005 g L-1, nanoaggregates seem to form only after DN exceeds 250%. A constant diameter of about 80 nm is obtained in the DN range of 100-300% for the solutions with 0.01 and 0.03 g L-1 polymer concentrations. It is noted in Figure 3a that particles with larger diameter exist in the lower DN region. This suggests that the aggregates are swollen with water because the repulsion between carboxylate anions still remains in the lower DN region. The dependence of the size of the aggregates on the polymer concentration is presented in Figure 3b for the sample with 100% DN. The size was unchanged for several weeks. The diameter of the aggregates is nearly 80 nm when the polymer concentration is above 0.01 g L-1 (Figure 3b). From this result, the cac is evaluated to be ∼0.01 g L-1 when the DN is 100%. 2. Polarity of the Interior of the Aggregate Particles. Fluorescence spectra and excitation spectra of Py (0.6 µM) in solutions with various concentrations of PEOb-PMA (100% DN) are shown in Figure 4. The feature of the fluorescence spectral change is that the intensity of the monomer emission increases with increasing PEOb-PMA concentration. This suggests that Py molecules were transferred from aqueous phase to the aggregates. The incorporation of nonpolar molecule Py into the aggregates implies that the polarity of the aggregates is lower than that of bulk aqueous phase. Additionally, we can note the significant shift of the excitation spectra. Similar shifts of the excitation spectra were reported by the Winnik group when Py was incorporated into the micelles of poly(styrene)-b-poly(ethylene oxide) in aqueous solutions.3,4 Therefore, it seems that the micropolarity of the aggregates is considerably different from that of the bulk phase.

Nanoaggregates of PEO-b-PMA Copolymer

Figure 4. Fluorescence spectra and excitation spectra of Py (0.6 µM) in the solutions of different concentrations of PEOb-PMA and CTAC. DN ) 100% in all solutions. λex ) 334 nm for part a; λem ) 392 nm for part b.

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Figure 5. (a) I1/I3 ratio of Py (0.6 µM) as a function of the degree of neutralization: C(PEO-b-PMA) ) 0.00 g L-1 (O), 0.005 g L-1 (b), 0.01 g L-1 (0), 0.03 g L-1 (9). (b) I1/I3 ratio of Py (0.6 µM) as a function of PEO-b-PMA concentration: DN ) 0% (O); 10% (b); 50% (0); 100% (9). The counterion was CTAC for both parts a and b.

To obtain insight into the micropolarity of the aggregates, we examined the vibronic fine structure of Py (the so-called I1/I3 ratio; see Figure 4a). It is known that the I1/I3 of Py can be used as a measure of the polarity of the environments of this probe.34 The higher value of I1/I3 indicates a higher polarity of the environments (i.e. rich in water) of the probe. The I1/I3 ratio is expected to decrease at the onset of the nanoaggregate formation, reflecting the preferential solubilizition of Py into a less polar microenvironment. Illustrated in Figure 5 is the dependence of I1/I3 on DN and the polymer concentration. In Figure 5a, the I1/I3 value decreases at first and becomes constant above DN ) 40% for the solutions with polymer concentration of 0.03 g L-1. Similar tendency is observed for the solutions with polymer concentrations of 0.005 and 0.01 g L-1, but the I1/I3 value becomes constant above 80%. In Figure 5b, as the polymer concentration is increased, a drastic decrease and then a leveling off in the I1/I3 value are noted around 0.01 g L-1 for the samples with DNs of 50% and 100%. Here it is clear from Figure 5 that the two factors (polymer concentration and DN) affect the nanoaggregate formation. Therefore, the aggregate parameters (cac, aggregation number, and so on) should be considered from the two aspects. Figure 5a gives a critical value of DN for every polymer concentration. When the polymer concentration is 0.03 g L-1, for example, DN should be at least 40% for the nanoaggregates to be formed. Figure 5b gives the cac for every DN. The cac is about 0.01 g L-1 for the samples with DNs of 50% and 100%. The cac is unclear for the sample with DN ) 10% (probably higher than 0.05 g L-1). The cac value for DN ) 100% agrees well with that obtained from DLS measurements (Figure 3).

It is interesting to compare the I1/I3 value in the aggregate solutions with that in neat solvents. The I1/I3 value in the aggregate solution converges to about 1.4 at higher polymer concentration or higher DN, as seen from Figure 5. This value (1.4) is close to the values obtained for acetic acid (1.37) and ethyl acetate (1.37).33 Therefore, Py molecules seem to reside in a milieu similar to that in acetic acid or ethyl acetate. 3. Viscosity of the Interior of the Aggregate Particles. Microviscosity is one of important parameters of micellar systems. In the case of low molecular weight surfactant micelles, the microviscosity has been investigated by measuring excimer formation of Py derivatives and fluorescence polarization of some suitable probe.33 In this study, we applied these techniques to the studies of the microviscosity of the aggregates from the CTAC/PEOb-PMA system. In Figure 4a, we note the excimer emission of Py ranging from 450 to 500 nm. The excimer emission is significant in the higher polymer concentration samples. It is wellknown that the Py excimer emission starts to appear when the Py concentration reaches about 1 mM in homogeneous solutions of nonviscous solvents such as methanol and hexane.35 Now there is excimer formation in the aggregate systems even though the total concentration is 0.6 µM. This suggests that Py molecules are highly concentrated into the aggregates. Therefore, the excimer emission seems to be an indicator for the formation of nanoaggregate. In Figure 6, we plot IE/IM vs polymer concentration, where IE and IM stand for the intensities of excimer fluorescence at 482 nm and monomer fluorescence at 392 nm, respectively. As the number of nanoaggregate molecules was increased, more Py molecules are transferred to the nanoaggregates. Then excimer formation becomes promi-

(34) Winnik, F. M.; Regismond, S. T. A. Colloids Surf. A: Physicochem. Eng. Aspects 1996, 118, 1.

(35) Turro, N. J. Modern Molecular Photochemistry; University Science Books: Mill Valley, CA, 1991.

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Figure 6. IE/IM of Py (0.6 µM) as a function of PEO-b-PMA concentration under different DN: (O) 10%, (b) 50%, (9) 100%. The counterion was CTAC.

Li and Nakashima

Figure 8. Degree of polarization of RB (5 µM) fluorescence as a function of DN in the nanoaggregates from PEO-b-PMA and CTAC. C(PEO-b-PMA) ) 0.01 g L-1 (b) and 0.03 g L-1 (O).

in the nanoaggregate particle to encounter each other within the lifetime of the excited state. Although the location of Py in the particle is unclear, the environment of the probe molecules is fluid to some extent. Fluorescence polarization also provides us with information on the viscosity of the aggregate particle (i.e. microviscosity), because the degree of polarization (P) is related with the microviscosity (ηm) around the probe by the Perrin-Weber equation

1/P ) 1/P0 + (1/P0 - 1/3)(kTτ/ηmV) Figure 7. Fluorescence excitation spectra of Py (0.6 µM) in the CTAC/PEO-b-PMA aggregates monitored at 392 nm (a) and 480 nm (b). C(PEO-b-PMA) ) 0.01 g L-1. DN ) 100%.

nent when more than one Py molecule is incorporated into single nanoaggregate particles. At much higher polymer concentration, the increased number of aggregates causes the repartion of Py molecules among the particles, resulting in the situation that the number of Py molecules in a single aggregate particle decreases. Thus, the excimer emission is decreased. It should be noted from Figure 6 that the maximum IE/IM value is positioned at lower polymer concentration if DN is higher. This agrees well with the conclusion from other observations that cac is lower when DN is higher. It is interesting to investigate the mechanism of excimer formation in the aggregate. Two types of mechanisms are considered: (a) static and (b) dynamic processes.36,37 The static process refers to the case where the excimer originates by direct excitation of the ground-state dimer. The dynamic process means that the excimer is formed by diffusive encounters of an excited monomer with a ground-state monomer. We can distinguish between the two cases by examining fluorescence excitation spectra and/or fluorescence decay curves. If the excimer forms through a dynamic mechanism, the excitation spectrum obtained by monitoring excimer emission (E-spectrum) coincides with that obtained by monitoring monomer emission (M-spectrum), whereas the E-spectrum shifts toward longer wavelength when the excimer forms via a static mechanism.36,37 In Figure 7, we show the M- and E-spectrum of Py in the aggregate. As seen from Figure 7, there is no difference in the band position in the M- and E-spectra. This indicates that the dominant mode of excimer formation is the dynamic process in the aggregate. From this fact we know that Py molecules can move around (36) Winnik, F. M. Chem. Rev. 1993, 93, 587. (37) Nakashima, K.; Kido, N.; Yekta, A.; Winnik, M. A. J. Photochem. Photobiol., A: 1997, 110, 207.

(3)

where P0 is the degree of polarization in rigid media, k the Boltzmann constant, T the absolute temperature, τ fluorescence lifetime, and V the effective volume of the probe.32 According to the eq 3, P becomes large with an increase in viscosity. We used RB as a probe for such purpose, because this probe seemed to be effectively incorporated into the PMA core of the aggregates due to the electrostatic interaction between the positive charge of this probe (see Figure 1) and the negative charge of the carboxylate ion in PMA block. In Figure 8, we plot P against DN for the CTAC/ PEO-b-PMA system for two polymer concentrations, 0.01 and 0.03 g L-1. In both cases, we note that P shows the maximum at DN ) 20%, and then its value gradually decreases with increasing DN, finally falling into constant values. The first increase in P seems to indicate the onset of the aggregate formation. As to the decrease in P after the maximum, there seem to be two possible reasons: (1) the lifetime of RB fluorescence increases with increasing DN (i.e. with increasing number of the aggregate particles), resulting in a decrease in P according to the Perrin-Weber equation, and (2) the effect of multiscattering of excitation and emission light on P becomes significant upon increasing the number of particles. Although the predominant effect on the change in P in Figure 8 is currently unclear, we know from Figure 8 that the viscosity of the aggregate is significantly higher than that of bulk aqueous phase. It is noted from Figure 8 that the average value of P is higher in the solution with higher polymer concentration. This seems to be ascribed to the fact that the fraction of RB in the aggregate particles increases with increasing polymer concentration. 4. Kinetics of Exchange of Incorporated Dyes between the Aggregate Particles. The kinetics of the exchange of the incorporated species between the aggregate particles is one of the important properties. To elucidate it, we designed an experiment using a fluorescence quenching technique. The idea is schematically shown in Figure 9. We prepared two kinds of aggregate solutions: one contains only R6G in the particles and the

Nanoaggregates of PEO-b-PMA Copolymer

Figure 9. Schematic representation for two model cases of the exchange of the incorporated dyes between the aggregate particles.

Figure 10. Time dependence of R6G fluorescence in the aggregates from the CTAC/PEO-b-PMA system. The concentration of PEO-b-PMA is 0.01 g L-1 and DN is 80% through the experiment. The concentration of R6G is 3 µM at point A. The concentration of R6G and MG in the end are 2 and 20 µM, respectively.

other only MG in the particles. It is known that the fluorescence of R6G is efficiently quenched by MG through electronic energy transfer.38-40 The Fo¨rster radius of the R6G-MG pair is known to be about 6 nm,39 which is much smaller than the radius of the aggregate particle (80-100 nm). Therefore, the energy transfer quenching of R6G fluorescence will effectively occur when R6G and MG are incorporated in the same particle, while it will not occur when R6G and MG are partitioned among different particles. We monitor the fluorescence intensity of R6G before and after the mixing of the two solutions. If the exchange of the incorporated dyes occurs on a second or minute time scale (the case B in Figure 9), we will observe a quick decrease in the fluorescence intensity. If it takes hours, days, or much longer time for the exchange to occur (the case A in Figure 9), we will not observe significant intensity change. Figure 10 shows the time profile of the fluorescence intensity in the aggregate solution. At point A, the sample contains only R6G. We quickly added the solution containing MG at point B and monitored the fluorescence intensity. The fluorescence intensity rapidly decreased to the level of point C. Line D represents the fluorescence (38) Nakashima, K.; Liu, Y. S.; Zhang, P.; Duhamel, J.; Feng, J.; Winnik, M. A. Langmuir 1993, 9, 2825. (39) Nakashima, K.; Duhamel, J.; Winnik, M. A. J. Phys. Chem. 1993, 97, 10702. (40) Tamai, N.; Yamazaki, T.; Yamazaki, I.; Mizuma, A.; Mataga, N. J. Phys. Chem. 1987, 91, 3503.

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intensity for the sample in which R6G and MG are premixed in the solution of the aggregates at the same concentrations as point C. It is confirmed from a control experiment that the rapid decrease in the fluorescence intensity to the level of point C is not due to the dilution of the initial sample with the solution containing MG.41 Therefore, we can conclude that the exchange of the incorporated dyes takes place within a time scale of seconds. The kinetics of exchange of the unimers between the aggregate particles is also interesting, because we can elucidate if the aggregates have dynamic or static nature. This can be examined by a method similar to that of Mattice et al.18 However, we need the block copolymers that are covalently labeled with fluorescence probes. Such an experiment will be done in a future work. Conclusions This study was oriented to investigate the properties of the nanoaggregtes of PEO-b-PMA copolymer formed by binding of CTAC to the PMA block through electrostatic attraction. The solubility behaviors of block copolymer complexes are quite different from those of homopolymer complexes. In contrast to the complexes of PMA homopolymer, which cause precipitation, the PEO-b-PMA complexes form water-soluble and stable particles with a size ranging from 80 nm to 200 nm. Light scattering and fluorescence measurements indicate that the cac is a function of both polymer concentration and DN. Therefore, the cac is estimated at each value of DN for the present system. Fluorescence probe techniques revealed that the probes employed are effectively incorporated into the aggregates in aqueous solutions. The vibronic fine structure of the monomer fluorescence of Py shows that Py molecules are incorporated into a domain that has polarity close to that of neat acetic acid or ethyl acetate. Fluorescence polarization of RB in the aggregate suggests that the microviscosity of the aggregates is higher than that of the aqueous phase. However, measurements of Py excimer formation provide us with the information that the aggregate is fluid to an extent that the excimer is formed via a diffusive collision mechanism. The kinetics of the exchange of the incorporated species between the aggregate particles was investigated by a fluorescence quenching technique. Cationic dyes, R6G and MG, were employed as the fluorophore and quencher, respectively, because it is known that the fluorescence of R6G is effectively quenched by MG through energy transfer from the former to the latter (Fo¨rster radius of about 6 nm). The aggregate particles loaded with R6G were quickly mixed with the particles loaded with MG in an aqueous solution, then the fluorescence intensity of R6G is monitored. From the time profile of the fluorescence intensity of R6G, we concluded that the exchange of R6G and MG between the aggregate particles takes place within a time scale of seconds. Acknowledgment. The authors thank Prof. Murata (Fukuoka University) for his valuable comments about the results of DLS measurements. The cost of the present work is partly defrayed by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (No. 12640559). LA0258852 (41) The concentration of R6G is 3 and 2 µM at points B and C, respectively. Therefore, the contribution of dilution to the intensity decrease is one-third of the original intensity (i.e. 120/3 ) 40). As seen in Figure 10, the decrease in intensity is about 90 ()120 - 30) on going from point B to C. The difference between the values 40 and 90 can be ascribed to the energy transfer quenching.