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Reversible Aggregation of Polystyrene-block-poly(2-vinylpyridine)-block-poly(ethylene oxide) Block Copolymer Micelles in Acidic Aqueous Solutions Miroslav Sˇ teˇpa´nek, Pavel Mateˇjı´cˇek, Jana Humpolı´cˇkova´, and Karel Procha´zka* Department of Physical and Macromolecular Chemistry and Laboratory of Specialty Polymers, School of Science, Charles University in Prague, Albertov 2030, 12840 Prague 2, Czech Republic Received June 21, 2005. In Final Form: August 10, 2005 Micelles of polystyrene-block-poly(2-vinylpyridine)-block-poly(ethylene oxide) (PS-PVP-PEO) were studied in acidic aqueous solutions by static and dynamic light scattering, alkalimetric titration, fluorescence correlation spectroscopy, and after deposition on a mica surface by atomic force microscopy. The PSPVP-PEO micelles prepared by dialysis in ternary 1,4-dioxane-methanol-acidic water mixtures have a very low association number and show a strong tendency to form aggregates. The aggregation, which is promoted at low pH, seems to be fully reversible. Possible mechanisms of the aggregation are discussed. Atomic force microscopy scans of PS-PVP-PEO micelles deposited on a mica surface reveal the formation of micellar aggregates and support the general concept of aggregation upon changes in conditions and deterioration of the stability of small micelles.
Introduction The key factor determining the solution behavior of poly(ethylene oxide) (PEO) is the presence of both a hydrophobic (-CH2CH2-) group and a hydrophilic (strongly hydrogen bonding) oxygen atom in its monomer unit. The amphiphilic character manifests itself by the strong aggregation tendency of PEO in different solvents. The presence of aggregated PEO chains in solutions has been evidenced in several studies by various experimental techniques including static1-4 and dynamic3-5 light scattering and small-angle neutron scattering6 or pulsed-fieldgradient NMR spectroscopy.3 Although different explanations of the aggregation mechanism, such as crystallization,1 interchain hydrogen bonding, or chain ends effect6 and subtle phase separation,7 have been proposed, the exact origin of PEO aggregation remains uncertain and may depend on experimental conditions including PEO concentration and temperature and also specific interactions with solvent molecules and other components. PEO plays the role of the shell-forming block in a number of self-assembling biocompatible systems designed for targeted drug delivery, such as popular poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (Pluronics),8 poly(ethylene oxide)-block-poly(L-lysine), poly(ethylene oxide)-block-poly(L-aspartic acid),9 poly(ethylene oxide)-block-poly(DL-lactide),10 and so forth, and * To whom correspondence should be addressed. E-mail:
[email protected]. (1) Polik, W. F.; Burchard, W. Macromolecules 1983, 16, 978. (2) Zhou, P.; Brown, W. Macromolecules 1990, 23, 1131. (3) Kinugasa, S.; Nakahara, H.; Fudagawa, N.; Koga, Y. Macromolecules 1994, 27, 6889. (4) Polverari, M.; van de Ven, T. G. M. J. Phys. Chem. 1996, 100, 13687. (5) Duval, M. Macromolecules 2000, 33, 7862. (6) Hammouda, B.; Ho, D. L.; Kline, S. Macromolecules 2004, 37, 6932. (7) De Gennes, P. G. C. R. Acad. Sci., Ser. II 1991, 313, 1117. (8) Kabanov, A. V.; Batrakova, E. V.; Alakhov, V. Y. J. Controlled Release 2002, 54, 159. (9) Lavasanifar, A.; Samuel, J.; Kwon, G. S. Adv. Drug. Delivery Rev. 2002, 82, 189.
in many other frequently studied water-soluble micellizing block copolymer systems, such as polystyrene-block-poly(ethylene oxide).11,12 The shells of multimolecular micelles represent highly concentrated (often incompletely hydrated) PEO domains, the limited solubility of which may be one possible cause contributing to the secondary aggregation of micelles with PEO shells observed in our study. Recently, we reported on the aggregation of micelles formed by polystyrene-block-poly(2-vinylpyridine)-blockpoly(ethylene oxide), PS-PVP-PEO, in aqueous solutions.13 The micellization behavior of this copolymer is strongly pH-dependent because of the presence of PVP, which is protonized and soluble in acidic solutions at pH lower than 4.8, whereas the deprotonized PVP that exists at higher pH is water-insoluble. The PS-PVP-PEO micelle is a three-layer nanoparticle in which PVP blocks form a middle layer between the compact PS core and the PEO shell. The PVP middle layer is either collapsed at high pH so that PS-PVP-PEO micelles resemble onion micelles formed by PS-PVP and PVP-PEO diblock copolymers or partially protonized, swollen, and reasonably flexible in acidic aqueous media so that the PVP layer becomes an inner shell between the core and the outer shell of PEO. Multimolecular PS-PVP-PEO micelles can be prepared by dialysis from 1,4-dioxane-methanol mixtures both in acidic and alkaline aqueous solutions. The micelles show a pronounced aggregation tendency, which is very strong in neutral and alkaline solutions and much more weakly pronounced in acidic solutions. (10) Liggins, R. T.; Burt, H. M. Adv. Drug. Delivery Rev. 2002, 54, 191. (11) Xu, R. L.; Winnik, M. A.; Hallett, F. R.; Riess, G.; Croucher, M. D. Macromolecules 1991, 24, 87. (12) Bronstein, L. M.; Chernyshov, D. M.; Vorontsov, E.; Timofeeva, G. I.; Dubrovina, L. V.; Valetsky, P. M.; Kazakov, S.; Khokhlov, A. R. J. Phys. Chem. B 2001, 105, 9077. (13) Sˇ teˇpa´nek, M.; Humpolı´cˇkova´, J.; Procha´zka, K.; Hof, M.; Tuzar, Z.; Sˇ pı´rkova´, M.; Wolff, T. Collect. Czech. Chem. Commun. 2003, 68, 2120.
10.1021/la0516680 CCC: $30.25 © 2005 American Chemical Society Published on Web 09/24/2005
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In our previous paper,13 we found that the micelles prepared by the titration of PS-PVP-PEO solution in a 1,4-dioxane-methanol mixture with 0.1 M HCl, followed by dialysis against 0.1 M HCl, had a lower molar mass than those prepared in alkaline solutions using 0.1 M NaOH. This difference is due to the fact that the PVP block is water-soluble below pH 4.8 and water-insoluble above pH 4.8. Hence the formation of PS-PVP-PEO micelles requires the association of a substantially lower number of copolymer chains in 0.1 M HCl than in 0.1 M NaOH. A transfer of micelles prepared in acidic solution to alkaline media is possible, but it is usually accompanied by the formation of water-soluble aggregates and sometime by partial precipitation. Several factors contribute to the destabilization of micelles after a pH increase: (i) The PVP blocks become insoluble, and the length of soluble shell-forming chains suddenly drops while the size and mass of insoluble cores increase. Because the micellar cores are almost frozen and the unimer exchange between micelles does not occur, the association number of original small micelles remains constant, which results in the decreased thermodynamic stability of the system. (ii) The micellar core is small, and the concentration of PEO chains is fairly high. When the PVP middle layer shrinks at pH higher than 4.8, PEO chains come closer to each other. The decrease in the volume of the shell worsens the problems of insufficient solvation of PEO blocks close to the core and contributes to the destabilization of micellar solutions. (iii) There is another possible cause of aggregation, which was suggested by a referee of this paper and which, after the critical reevaluation of all data, seems to be highly probable in this particular micellar system for the following reason. The preparation protocol (based on dialysis in ternary 1,4-dioxane-methanol-acidic water mixtures) that was used results in micelles with a very low association number. The micellization equilibrium and molar mass of micelles actually stop to change (see later) in mixtures containing ca. 45 vol % of both 1,4dioxane and methanol and 10 vol % of acidic H2O. During further dialysis in acidic aqueous media, the PS cores shrink and become fairly compact. However, because the cores are very small, they are probably not glassy in aqueous media. This assumption may be supported by the following arguments. It has been proven experimentally that the Tg of thin polymer films is lower that that of corresponding bulk polymers.14 Furthermore, we found in our previous studies that PS cores of small PS-PVPPEO micelles are soft and deform (flatten) after deposition on mica13 whereas larger PS cores of PS-PMA micelles deposited on mica are hard and do not deform.15 Hence we assume that PS blocks in cores are not kinetically frozen and may reorganize rather than that they are relatively short and are not firmly entangled. In this case, any destabilization of micellar systems may cause at least a partial reorganization of micellar cores and the formation of more stable aggregated nanoparticles. This mechanism is supported by another observation. Micelles formed by the same copolymer in aqueous systems were also studied by Jerome et al.16 The authors used another preparation protocol resulting in a higher association number, and they do not mention any aggregation at all. (14) Bliznyuk, V. N.; Assender, H. E.; Briggs, G. A. D. Macromolecules 2002, 35, 6613. (15) Mateˇjı´cˇek, P.; Humpolı´cˇkova´, J.; Procha´zka, K.; Tuzar, Z.; Sˇ pı´rkova´, M.; Hof, M. Webber, S. E. J. Phys. Chem. B 2003, 107, 8232. (16) Lei, L. C.; Gohy, J. F.; Willet, N.; Zhang, J. X.; Varshney, S.; Jerome, R. Macromolecules 2004, 37, 1089.
In this article, we study the pH-provoked aggregation of PS-PVP-PEO micelles in acidic solutions. We propose a possible aggregation mechanism, address the role of reorganization processes in PS cores, the protonization of the PVP layer and the solubility of the PEO outer layer of small micelles in the aggregation process, and discuss the effect of HCl concentration on the formation of aggregates. To support the idea of the formation of large compound micelles after the destabilization of the original small micelles, we performed an AFM study of the aggregation after the deposition of micelles on the hydrophilic mica surface and compared the aggregation at the surface with that in solutions. Experimental Section Materials. Copolymer Sample. The triblock copolymer polystyrene-block-poly(2-vinylpyridine)-block-poly(ethylene oxide) (PS-PVP-PEO) was purchased from Polymer Source, Ltd. (Dorval, Quebec, Canada) and used as obtained. The numberaveraged molar masses of PS, PVP, and PEO blocks and the polydispersity index of the sample, according to the data provided by the manufacturer, are 1.41 × 104, 1.23 × 104, and 3.50 × 104 g/mol and 1.08, respectively. Preparation of Micelles. PS-PVP-PEO micelles were prepared according to the following protocol: The sample was dissolved in a 1,4-dioxane (80 vol %)-methanol mixture overnight. After full dissolution, methanol was slowly added drop by drop under very mild stirring until 50% methanol content was reached. Then a 0.01 M HCl aqueous solution was slowly added until 50% water content was reached. The next step consisted of the dialysis of the solution against 0.01 M HCl, followed by dialysis against an HCl solution of desired concentration. During the final dialysis, the HCl solution in the external bath was exchanged several times to ensure the removal of all traces of organic solvents. The concentration of polymers before the dialysis was 3 g/L. The exact final polymer concentration was determined from volume changes during dialysis. Reagents and Solvents. Octadecylrhodamine B (ORB) was purchased from Molecular Probes (Eugene, OR). Spectrophotometric-grade solvents (Aldrich) and deionized water were used in the study. Techniques. Light Scattering. The setup, used for both static (SLS) and dynamic (DLS) light scattering measurements (ALV, Langen, Germany), consists of a 633 nm He-Ne laser, an ALV CGS/8F goniometer, an ALV High QE APD detector, and an ALV 5000/EPP multibit, multitau autocorrelator. The solutions for measurements were filtered through 0.45 µm Acrodisc filters. The measurements were carried out for different concentrations (0.1-0.75 g/L) and different angles at 20°C. The measurements for the low ionic strength solutions were performed in quartz cells. The SLS data were treated by the standard Zimm method using the equation
1 Kc ) + 2A2c Rcor(q, c) P(q)Mw
(1)
where K ) 4πn2(dn/dc)2/(λ4NA) is a constant containing the refractive index of the solvent, n, refractive index increment of the polymer with respect to the solvent, (dn/dc), wavelength of the incident light, l, and Avogadro’s constant, NA. Rcor(q, c) is the corrected Rayleigh ratio, which depends on the polymer concentration c and on the magnitude of the scattering vector, q ) (4πn/λ)sin(ϑ/2), where ϑ is the scattering angle, Mw is the apparent weight-average molar mass of scattering polymeric particles, A2 is the “light-scattering-weighted” second virial coefficient of the concentration expansion, and P(q) is the particle form factor that accounts for intraparticle interference effects. To evaluate the measurements, we assumed that the relationship P-1(q) ) 1 + 1/ R 2q2 is fulfilled in the region of reasonably low q irrespective 3 g of the particle shape. The treatment yields either “true” zaveraged radii of gyration, Rg (i.e., the values obtained by the extrapolation to zero concentration) or concentration-dependent “apparent” radii of gyration, (Rg)app. The refractive index incre-
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ment of the copolymer in water, (dn/dc) ) 0.190, was calculated as a weight-average value corresponding to the copolymer composition and the literature data17 for the increments of the individual homopolymers in water. The DLS data analysis was performed by fitting the field autocorrelation function g1(t), related to the measured normalized intensity autocorrelation function g2(t), by the Siegert relation g2(t) ) 1 + β[g1(t)]2, where β is a factor accounting for deviation from the ideal correlation. An inverse Laplace transform of the g1(t) function with the aid of a constrained regularization algorithm (REPES) provides the distribution of relaxation times, τA(τ)
g1(t) )
∫
∞
-∞
( τt) d ln τ
τA(τ) exp -
(2)
Diffusion coefficients were calculated from mean relaxation times, 〈τ〉, of individual modes as D ) 1/〈τ〉q2 in dilute solutions (copolymer concentration, c ) 0.09 g/L) where interparticle interactions may be neglected. Hydrodynamic radii, RH, were evaluated from the diffusion coefficients using the StokesEinstein equation. Fluorescence Correlation Spectroscopy (FCS). FCS measurements were performed with a ConfoCor I (Carl Zeiss, Germany) binocular microscope equipped with a 514 nm argon laser, an adjustable pinhole together with special fluorescence optics, an SPCM-200PQ detection diode, and an ALV-5000 correlator (ALV, Langen, Germany). Micelles were noncovalently labeled by an amphiphilic fluorescent probe, octadecylrhodamine B (ORB). The measured normalized autocorrelation functions, G(t), of fluctuating fluorescence intensity, F(t), given by the equation18
G(t) ) 1 +
〈F(0) F(t)〉 〈F(0)〉
2
(3)
have been used for the evaluation of the average number of fluorescent particles, N, in the irradiated volume.18,19 (According to current experimental conditions, the irradiated volume was ca. 10-16 L.) Number-average molar masses, Mn, have been calculated from N, as described in our earlier paper.15 Atomic Force Microscopy (AFM). AFM measurements were performed in tapping mode under ambient conditions using a commercial scanning probe microscope, Digital Instruments NanoScope dimensions 3, equipped with a Nanosensors silicon cantilever with a typical spring constant of 40 N m-1. Details of the measurement and the principle of evaluation of the micellar polydispersity were given in our earlier paper.15 Polymeric micelles were deposited on a fresh (i.e., freshly peeled off) mica surface (muskovite, theoretical formula KAl2(AlSi3O10)(F,OH)2, Geological Collection of Charles University in Prague, Czech Republic) by a fast dip coating in a dilute micelle solution in 10-4-10-1 M HCl (copolymer concentration ca. 10-2 g/L). After the evaporation of water, the samples for AFM were dried in a vacuum oven at ambient temperature for ca. 5 h. Potentiometry. The pH measurements were performed with a Radiometer PHM 93 reference pH meter equipped with a PHC 3006 combined glass microelectrode.
Results and Discussion Characterization of Nonaggregated Small PSPVP-PEO Micelles by Light Scattering and Fluorescence Correlation Spectroscopy. At first, we addressed the questions of reversibility of the micellization equilibrium and changes in the molar mass of micelles during the dialysis. We measured DLS from two solutions with fairly similar solvent compositions (before and after the scattering intensity of unimers drops to zero). The (17) Polymer Handbook, 3rd ed.; Brandup, J., Immergut, E. H., Eds.; Wiley-Interscience: New York, 1989; Vol. VII, pp 409-484. (18) 18) Webb, W. E. In Fluorescence Correlation Spectroscopy Theory and Applications; Riedler, R., Elson, E. S., Eds.; Springer-Verlag: Berlin, 2001. (19) Hink, M. A.; van Hoek, A.; Visser, A. J. W. G. Langmuir 1999, 15, 992.
Figure 1. Relaxation time distributions for PS-PVP-PEO solutions (copolymer concentration c ) 0.75 g/L, scattering angle ϑ ) 90°) in solutions of 1,4-dioxane(50 vol %)-methanol mixture (curve 1), 1,4-dioxane(45 vol %)-methanol(45 vol %)-0.01 M HCl (curve 2), and 10-4 M HCl (curve 3). Table 1. Molar Mass, Mw, Radius of Gyration, Rg, and Second Virial Coefficient, A2, of PS-PVP-PEO Nanoparticles in 10-1 and 10-4 M HCl Solutions solution
Mw × 10-6 g/mol
Rg nm
A2 × 107 mol L/g2
10-1 M HCl 10-4 M HCl
9.33 0.71
142.1 25.5
1.28 2.45
results are shown in Figure 1. It is evident that while in the binary 1,4-dioxane-methanol (50 vol %) mixture (curve 1) the micelles (slow mode) coexist in equilibrium with unimers (fast mode), after the addition of 10 vol % of acidic water (curve 2) the unimer contribution to the scattering is no longer measurable. The micellar peak is rather broad (slightly narrower in 10-4 M HCl than in 10-2 M HCl), but it sharpens after the transition in acidic water (see later text). Second, we characterized micelles prepared by dialysis in aqueous HCl by static and dynamic light scattering. Because our previous results showed that the tendency of PS-PVP-PEO micelles to aggregate in acidic solution strongly increases with increasing ionic strength, we performed the light scattering determination of weightaverage molar masses in fairly dilute, low ionic strength HCl solutions (without added salt) at pH 4 where the partial protonation of PVP blocks is small (the Zimm plot is regular and intermicellar interactions are negligibles see later text) but it prevents or at least hinders possible aggregation. The characteristics are given in Table 1. A very important feature of the aqueous micellar system with very little protonated PVP at pH 4 is that its fairly dilute solutions do not contain micellar aggregates (compound micelles). Because aggregation increases with concentration and LS characterization requires (i) a nonnegligible range of concentrations and (ii) yields either weigh-average or z-average data (strongly affected even by traces of aggregates), we also applied fluorescence correlation spectroscopy because it reliably works at very low polymer concentrations and provides number-average data. We wanted to determine if small nonaggregated micelles exist at high dilutions in a broader pH region. The numberaverage molar masses, Mn, of PS-PVP-PEO micelles in the pH range from 2 to 4 were determined by titrating dilute aqueous solutions of micelles with octadecyl-
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Figure 2. Fluorescent particle number, N, as a function of fluorescent probe (octadecylrhodamine B) concentration, cORB, obtained from an FCS measurement with a 0.01 g L-1 solution of PS-PVP-PEO micelles in 10-2 M HCl.
rhodamine B (ORB). The rate of fluctuation in fluorescence intensity from a small irradiated volume (proportional to the number of fluorescent particles in this volume), N, is measured by FCS, and Mn can be calculated as described in our earlier papers.15 Figure 2 shows the number of fluorescent particles in the irradiated volume, N, as a function of ORB concentration, cORB, for a PS-PVP-PEO solution in 10-2 M HCl (the curves for other solutions were similar). The measurements in the pH region of 2-4 yields pH-independent number-average molar mass of small micelles, Mn ) 0.8 × 106 g/mol. The FCS measurements indicate that no micellar aggregates form in very dilute (i.e., 10-2 g/L) acidic aqueous solutions of PS-PVPPEO. Light Scattering Study of the Aggregation of Micelles in Acidic Aqueous Solutions. We focused our new detailed study on PS-PVP-PEO behavior on acidic solutions for the following reasons: (i) In the high-pH region, the aggregation does not often reach the equilibrium state in a reasonable time, and the solutions undergo partial precipitation over several weeks. Hence we did not manage to obtain fully reproducible molar masses and sizes of aggregates in neutral and alkaline solutions in our previous study.13 (ii) At high pH, the rates and extents of aggregation are sensitive to mechanical shear, and all manipulations of solutions, such as stirring, shaking, or even filtration by membrane microfilters, resulted in a massive aggregation. Neutral and alkaline onion micelles are surface-active, and their solutions are strongly foaming. Therefore, the shaking-induced aggregation could be attributed to the aggregation of micelles adsorbed at the air-water interface of small air bubbles. We studied the behavior of micelles prepared in saltfree aqueous HCl solutions as a function of HCl concentration. We present all results of SLS measurements in the form of Zimm plots because it is a legitimate way of presenting SLS data and it immediately reveals any irregularity or complexity in the behavior. We also tried other types of plots, but they show no new features. Whereas in 10-1 and 10-4 M HCl the Zimm plots are fairly regular (not shown) and the concentration and angular dependences of light scattering data may be successfully treated using the second virial coefficient, A2, and the apparent radius of gyration, (Rg)app, in 10-2 and 10-3 M HCl they show a contribution of light scattering from large particles in the low-angle region, which increases with polymer concentration. (See the Zimm plot for the 10-3 M HCl solution in Figure 3.) Therefore, the evaluation of light scattering data by the standard Zimm technique, yielding the apparent weight-average molar mass, Mw,
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Figure 3. Zimm plot of the light scattering from PS-PVPPEO solutions in 10-3 M HCl. Plot constant k is 2000 L/g µm2.
apparent radius of gyration, (Rg)app, and second virial coefficient, A2, could have been carried out only for the PS-PVP-PEO solutions in 10-1 and 10-4 M HCl. The results are given in Table 1. At pH 4, we found a surprisingly low value (which corresponds to nonaggregated micelles), Mw ) 7.1 × 105 g/mol (i.e., the association number is only ca. 12). However, at pH 1 the apparent weight-average molar mass, Mw, is more than 10 times higher. We performed a careful study of changes in the apparent molar masses of micelles prepared under defined conditions (i.e., by dialysis in 0.01 M HCl) during their transfer, using stepwise dialysis, in another acidic medium (all experiments below pH 4.5) and back, and we found that the observed changes are reversible (i.e., the micelles aggregate and the aggregates decompose reversibly depending on HCl concentration). We repeated the transfer several times, and we got always the same Zimm plots for respective pH values of 1, 2, 3, and 4 (within the range of experimental errors). Even though the observed aggregation occurs in aqueous solutions, the small core size does not guarantee the glassy state of PS, as pointed out by a referee of this article. Therefore, we cannot preclude the facts that a secondary aggregation of micelles and the formation of a fraction of compound micelles with rearranged PS chains in cores of original micelles occur at pH 2 and 3, when PVP is charged and electrostatic interactions are not yet screened by excess H3O+ and Cl- ions. An increased electrostatic repulsion may produce a pull on the chains (as suggested by a referee), which would result in the formation of more stable, possibly nonspherical aggregated structures. Hence, we assume the following aggregation scenario: Even though the PS is strongly hydrophobic and the exchange of individual unimers between micelles in the aqueous phase is negligible, the PS chains in the cores are sufficiently flexible and mobile. They may reorganize upon changed conditions, and transient “hemimicelles” may stick together, forming large compound micelles. We depicted the proposed mechanism of formation and a possible structure of such compound micelles or aggregates with partially reorganized cores in Scheme 1. We believe that the observed behavior is caused by a combination of all of the above-described causes, but the reorganization of the cores seems to be a necessary precondition for the aggregation. The apparent radii of gyration, (Rg)app, evaluated from the initial slopes of the angular dependences of light scattering data, are plotted as functions of pH in Figure 4 for two finite PS-PVP-PEO concentrations. The Figure shows that the balance between small PS-PVP-PEO micelles and aggregates (or compound micelles) shifts in
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favor of aggregates (i) with increasing HCl and copolymer concentrations. The inset in Figure 4 shows the concentration dependence of (Rg)app in 10-3 M HCl. When discussing the apparent radii of gyration, it is necessary to keep in mind that they represent the z-average values and are strongly affected by the presence of large particles and overestimate their contribution. The interpretation of SLS data, based on the assumption of secondary aggregation and formation of compound micelles, is supported by DLS measurements. The distribution of relaxation times generally contains two peaks (Figure 5a and b). Angular dependences (Figure 5c) prove that both relaxation times correspond to diffusioncontrolled modes. The intensity of the slow mode decreases with increasing pH and decreasing copolymer concentration, and the slow mode disappears completely at very low copolymer and HCl concentrations. The dynamic light scattering measurements thus show that PS-PVP-PEO solutions contain either single micelles at low polymer and HCl concentrations or mixtures of single micelles (fast mode) and micellar aggregates (slow mode) at higher copolymer and HCl concentrations. The hydrodynamic radius of small (original) micelles, RHmic, calculated from the short relaxation time is plotted in Figure 6 (curve 1) as a function of pH. At low polymer concentrations (Figures 5a and 6), the changes in the position of the fast relaxation peak reflect the size changes of nonaggregated micelles due to electrostatic repulsion-to-screening interplay. Above pH 3, RH decreases with increasing pH as a result of substantial deprotonation of PVP blocks. A fairly pronounced maximum is reached around pH 3, and below this pH value, a decrease in RH with increasing concentration of the acid can be attributed to the screening of the
Figure 4. Apparent radius of gyration, (Rg)app, of PS-PVPPEO nanoparticles in HCl aqueous solutions as a function of pH. Copolymer concentrations are 0.09 (curve 1) and 0.75 g/L (curve 2). (Inset) (Rg)app as a function of copolymer concentration, c, in a 10-3 M HCl solution.
electrostatic repulsion between charged PVP segments by H3O+ and Cl- ions present in the shell. The sizes of aggregates are influenced by a complex interplay of electrostatic forces. They reflect both the pH-dependent changes in the aggregation number and the expansion of PVP blocks. The hydrodynamic radii of apparently polydisperse aggregates in 10-1 and 10-2 M HCl, based on the z average of diffusion coefficients, are 148 and 176 nm, respectively. Concerning the measurements for different polymer concentrations at constant pH 3, the changes in relaxation times are probably caused by intermicellar interactions. We observed analogous electrostatic interactions (and studied in detail their effects on SLS and DLS data) in systems of polystyrene-block-poly(methacrylic acidic) mi-
Figure 5. Relaxation time distributions for PS-PVP-PEO solutions in HCl aqueous solutions measured at scattering angle ϑ ) 90° for (a) copolymer concentration c ) 0.09 g/L in 10-4 (curve 1), 10-3 (curve 2), 10-2 (curve 3), and 10-1 M HCl (curve 4) and (b) in 10-3 M HCl for copolymer concentrations 0.09 (curve 1), 0.19 (curve 2), 0.38 (curve 3), and 0.75 g/L (curve 4). (c) Reciprocal mean relaxation time, 〈τ〉-1, of the slow (curve 1) and fast (curve 2) modes for a 0.75 g/L solution in 10-1 M HCl as a function of q2.
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Figure 6. Hydrodynamic radius, RHmic (curve 1), and degree of protonization of PVP blocks, R (curve 2), of nonaggregated PS-PVP-PEO micelles in HCl solutions as a function of pH. (RHmic was measured for copolymer concentration c ) 0.09 g/L at scattering angle ϑ ) 90°.)
celles in low ionic strength solutions20 and in systems of “hairy” PS-PVP heteroarm copolymers.21 We have shown that small ions escape from the shell (which otherwise behaves as a convex polyelectrolyte brush22) under certain conditions (at a fairly low degree of ionization and low ionic strength) and individual charged micelles interact over long distances. For a correct assessment of the fraction of micellar aggregates, it is necessary to take into account that the distribution of relaxation times is weighted by intensity and that the fraction of aggregates weighted by mass or by number is substantially lower. (Assuming the form factor of hard spheres for both the micelles and the aggregates, the mass fraction of aggregates for the 0.75 g/L solution at pH 3 is only about 0.09.) It is evident that the reversible aggregation of PSPVP-PEO micelles in acidic solutions is affected by the electrostatic repulsion between protonized PVP blocks. However, the electrostatic influence is quite complex. The Coulomb forces repel original micelles from each other, which prevents aggregation. The intramicellar interchain repulsion simultaneously produces a pull on protonized PVP blocks of micelle-forming chains that may cause a reorganization of PS in micellar cores, which facilitates the formation of compound micelles. The final effect is a result of the competition between the direct electrostatic forces between macroions and (short-range, mediumrange, and long-range) electrostatic screening by small ions. Repulsive forces and the consequent pull on PVP blocks increase with the degree of protonation of the PVP block, RPVP, but they are always, at least partially, screened by small ions. The value of RPVP, which can be determined by alkalimetric titration,21 is plotted in Figure 6 (curve 2) as a function of pH. The protonation of PVP blocks in PS-PVP-PEO micelles is apparently suppressed by the presence of PEO blocks, which form the outer shell of micelles and hinder the hydration of PVP. At pH 1, RPVP is only 0.48 (i.e., substantially lower than the value RPVP ) 0.65 found in our recent paper21 for PS/PVP star copolymer, where PVP blocks are in direct contact with the solvent). The addition of HCl increases the protonation of PVP blocks, but it very efficiently screens the electro(20) Mateˇjı´cˇek, P.; Podha´jecka´, K.; Humpolı´cˇkova´, J.; Uhlı´k, F.; Jelı´nek, K.; Limpouchova´, Z.; Procha´zka, K.; Sˇ pı´rkova´, M. Macromolecules 2004, 37, 10141. (21) Sˇ teˇpa´nek, M.; Mateˇjı´cˇek, P.; Humpolı´cˇkova´, J.; Havra´nkova´, J.; Podha´jecka´, K.; Sˇ pı´rkova´, M.; Tuzar, Z.; Tsitsilianis, C.; Procha´zka, K. Polymer, in press. (22) Misra, S.; Mattice W. L.; Napper D. H. Macromolecules 1994, 27, 7090.
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static repulsion. We may conclude that the aggregation is generally promoted by the addition of HCl. In this article, we studied mainly the effect of acidity (HCl) because we were interested in the behavior of micelles at low ionic strength when the electrostatic forces are screened very little and therefore are important. Because the addition of HCl results in changes in ionic strength, we discuss the effect of ionic strength throughout the article. We did more studies on the added salt effect in our previous paper.13 Nevertheless, in this work we did some measurements with added NaCl at pH 2 and 4. The addition of the salt suppresses the protonization of PVP very little, but it efficiently screens electrostatic interactions. In systems with PEO blocks, it also lowers the LCST of PEO and deteriorates the solubility and stability of small nonaggregated micelles with PEO shells.23 We found that increasing concentrations of NaCl promote aggregation both at pH 2 and pH 4. Because SLS data indicate that at an ionic strength about 10-4 mol/L electrostatic interactions are very weak because of a low degree of PVP protonization, we assume that the aggregation at pH 4 is a result of the lower solubility of PEO in salted solutions rather than a result of electrostatic screening. At low pH, an additional contribution to the aggregation mechanism should be taken into account. Reliable experimental data published by several research groups show that PEO chains form hydrogen-bond-stabilized interpolymer complexes (e.g., with neutral poly(acrylic acid) at low pH) in which PEO acts as a proton acceptor.24,25 The stability of these complexes is fairly high and is sufficient to stabilize layer-by-layer (LbL) films26 (by analogy to electrostatically stabilized LbL films).27 Because it is known that the pyridinium cation can act as a proton donor (more precisely, as a donor of partially positively charged bound hydrogen) and form hydrogen bonds,28 it is possible that the ends of partially protonized PVP blocks form hydrogen-bond-stabilized complexes with parts of PEO chains. Thus parts of both PEO and PVP chains could be immobilized in the complex, and the effective length of mobile PEO chains that ensure the solubility of micelles would further decrease. We have observed a similar immobilization of PEO chains in hydrogen-bondstabilized interpolymer complexes between PEO and poly(methacrylic acid) (PMA) in mixed shells of PS-PEO and PS-PMA micelles at low pH.29 Although the complex formation was not detected in dilute solutions of linear PVP-PEO copolymers at low pH, the difference in behavior of PVP-PEO and PS-PVP-PEO copolymers can be rationalized because linear chains with both blocks soluble at low pH form expanded flexible coils with a low concentration of monomer units whereas in micellar shells complex formation is promoted by the reduced effective flexibility of concentrated shell-forming blocks and by the fact that the degree of ionization increases in the radial direction from the core and reaches a maximum at the PVP/PEO “interface”.30 Recently, it was pointed out by Messina et al. that the formation of LbL films requires (23) Saeki, S.; Kuwahara N.; Nakata, M.; Kaneko, M. Polymer 1977, 18, 1027. (24) Pradip; Maltesh, C.; Somasundaran, P.; Kulkarni, R. A.; Gundiah, S. Langmuir 1991, 7, 2108. (25) Heyward, J. J.; Ghiggino, K. P. Macromolecules 1989, 22, 1159. (26) Sukhishvili, S. A.; Granick, S. Macromolecules 2002, 35, 301. (27) Lvov, Y.; Decher, G.; Mohwald, H. Langmuir 1993, 9, 481. (28) Bian, L. J. Phys. Chem. A 2003, 107, 11517. (29) Podha´jecka´, K.; Sˇ teˇpa´nek, M.; Procha´zka, K.; Brown, W. Langmuir 2001, 17, 4245. (30) Uhlı´k, F.; Limpouchova´, Z.; Jelı´nek, K.; Procha´zka, K. J. Chem. Phys. 2004, 121, 2367.
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Figure 7. AFM scan (1× 1 µm2, top view) of PS-PVP-PEO micelles deposited on a mica surface from (a) 10-1 M and (b) 10-2 M HCl solutions.
the contribution of other (nonelectrostatic or noncomplexforming, i.e., van der Waals) forces to immobilize the chains.31 On the basis of our light scattering study, we believe that the observed reversible aggregation of PS-PVPPEO micelles in acidic aqueous solutions is caused by a combination of all of the above effects and that a partial reorganization of PS chains in small and soft cores is a precondition and the main reason for the observed complex (apparently reversible) behavior. Atomic Force Microscopy Study of Micelles at the Mica Surface. To get supplementary information on the aggregation processes, we studied structural changes of micelles after their deposition on the hydrophilic mica surface by AFM. For atomic force microscopy studies, the PS-PVP-PEO nanoparticles were deposited from 10-410-1 M aqueous HCl solutions on a freshly peeled-off mica surface. Earlier we found that particle sizes of nonaggregating micelles evaluated from AFM scans are proportional to those measured in the solution by light scattering or fluorescence correlation spectroscopy, although the deposited particles are pancake-deformed because of the interaction of water-soluble shell-forming chains with the hydrophilic surface.15 Therefore, by scanning a sufficiently large number of micelles, both the weight- and number-average distributions of molar masses of studied nanoparticles can be determined. Figure 7 shows the top views of two 1 × 1 µm2 scans of PS-PVP-PEO micelles deposited on a freshly peeled-off mica surface from aqueous solutions: (a) in 10-1 M and (b) in 10-2 M HCl. The scan of mica dipped in a 10-1 M HCl solution of PS-PVP-PEO micelles (Figure 7a) shows micellar clusters, whereas micelles deposited from 10-2 (Figure 7b), 10-3, and 10-4 M HCl (not shown) show wellseparated micelles. The section analyses of both separated and aggregated (interconnected) micelles on the mica surface are shown in Figure 8. The centers of aggregated micelles are fairly well resolved, which is compatible with the concept of compound micelles with partially reorganized cores that stick together. The number-average distribution function of molar masses of deposited PSPVP-PEO micelles is shown in Figure 9. When comparing LS and AFM results, one has to be careful. At first, the absolute sizes of micelles change after their deposition and drying. The size is evaluated through interaction with the tip, and its finite size and possible (31) Messina, R.; Holm, C.; Kremer, K. Langmuir 2003, 19, 4473.
Figure 8. Typical results of a section analysis (graphs showing horizontal profiles during the straight line motion of the AFM tip over a deposited nanoparticle) of AFM scans shown in Figure 7: a cluster of micelles deposited from a 10-1 M HCl solution (curve 1) and a single micelle deposited from a 10-2 M HCl solution (curve 2).
Figure 9. Number-average distribution function of the molar masses of PS-PVP-PEO micelles deposited on a mica surface from a 10-2 M HCl solution.
specific interactions may affect the obtained values. Second, a lower concentration limit for light scattering measurements with PS-PVP-PEO micelles is about 0.1 g/L. Below this limit, the scattering intensity is too weak and does not provide reliable experimental results. However, this concentration is still too high for AFM measurements because it leads to mica surface compactly covered by the polymer. In our measurements, mica was dipped in a 10-2 g/L PS-PVP-PEO solution, which contains a considerably lower number of aggregates than micelles. Third, the aggregation mechanism and mainly the conditions supporting aggregation differ at the surface
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and in solutions. The fact that the number of aggregates on the surface is fairly high suggests that the aggregates observed by AFM have not been deposited from the solution. Instead, they form in situ at the wet mica surface probably because the local conditions change and the forces acting at the surface are different from those in solutions. This is why a direct and fully conclusive comparison of AFM and light scattering data is impossible for the studied system. However, we would like to stress that we are not interested in a direct comparison of SLS and DLS with AFM data. We look for additional pieces of information supporting the idea of the formation of compound micelles in the studied system, and we believe that the AFM study contributes to the understanding of the aggregation process. The destabilization of the solution and the certain pull of micelle-forming chains due to the spreading of their soluble blocks on the hydrophilic mica surface promote the aggregation. This observation is indirect, but it is compatible with the basic concept of the formation of compound micelles. Furthermore, the AFM study shows that the deposition of micelles from solutions containing lower HCl concentrations results in well-separated micelles, whereas the surface-induced aggregation occurs after the deposition of solutions containing higher concentrations of HCl. We can generally conclude that an enhanced aggregation tendency of PS-PVP-PEO micelles at low pH has been observed both in solutions and at the surface. Conclusions Light scattering, fluorescence correlation spectroscopy, and atomic force microscopy of triblock copolymer polystyrene-block-poly(2-vinylpyridine)-block-poly(ethylene oxide) in aqueous solutions containing 10-4-10-1 M hydrochloric acid show that PS-PVP-PEO micelles (Mw of ca. 0.7 × 106 g/mol and RH of ca. 30 nm) aggregate and form ca. 150 nm clusters at low pH. As compared with our recent study in neutral and alkaline solutions, the aggregation tendency is lower; however, it is still important (depending on the conditions). The aggregation seems to be reversible
and occurs spontaneously with changing external conditions. The average cluster size and weight fraction of aggregates strongly vary with polymer, HCl, and salt concentrations. All data indicate that the observed intermicellar aggregation occurs as a complex result of a combination of several effects. Even though we performed extensive studies of aqueous solutions of PS-PVP-PEO micelles, supplementary research (using other techniques) will be needed to present an unambiguous explanation of the aggregation mechanism. At the present stage, we believe that small PS cores of micelles (prepared by dialysis in the 1,4-dioxane-methanol-acidic water ternary mixture) are not glassy and some reorganization of PS chains in cores occurs and facilitates the secondary reversible aggregation of small micelles. Atomic force microscopy measurements on PS-PVPPEO micelles deposited on fresh mica surfaces by fast dip coating revealed an in situ formation of micellar clusters from very dilute solutions that contain only negligible amounts of aggregates (as confirmed both by LS and FCS measurements). Even though it is impossible to compare the results of solution studies and AFM directly, both techniques show a strong aggregation tendency of PSPVP-PEO micelles and prove that the aggregation increases with increasing concentrations of HCl. Acknowledgment. M.Sˇ . and K.P. acknowledge the financial support of the Grant Agency of the Czech Republic (grant nos. 203/02/D048 and 203/04/0490, respectively). We thank the Marie Curie Research and Training Network (grant no. 505 027, POLYAMPHI) for support. We are indebted to the referees of this article for their constructive comments and suggestions and particularly for an alternative explanation of the observed aggregation tendency, which after a critical reevaluation of existing experimental data together with some supplementary measurements seems to be the most probable cause of the observed complex behavior. LA0516680
