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Formation of Micelles with Complex Coacervate Cores M. A. Cohen Stuart,* N. A. M. Besseling, and R. G. Fokkink Laboratory for Physical Chemistry and Colloid Science, Wageningen Agricultural University, P.O. Box 8038, 6700 EK Wageningen, The Netherlands Received June 29, 1998. In Final Form: September 7, 1998 Micelles are commonly regarded as colloidal structures spontaneously formed by amphiphilic molecules, that is, molecules consisting of two distinct parts of which one is soluble and the other is insoluble. This definition is too restrictive: other kinds of molecules can also form micelles. We report on the formation of micelles from a mixture of a (water-soluble) polyanion and a diblock copolymer with two entirely watersoluble blocks: one cationic and one neutral. The cationic block forms a complex coacervate with poly(acrylic acid); the neutral block serves as a stablizing block, prohibiting the growth of the complex coacervate droplets to macroscopic sizes. The formation of these micelles upon mixing is preceded by a macroscopic phase separation. The polymer-rich phase which initially forms rearranges into a stable micellar solution.
Introduction There are vast amounts of literature on solutions of amphiphilic molecules.1 Since the word “micelle” was coined early in this century2 to indicate the tiny selfassembled structures formed by surfactants, the study of structures formed by amphiphilic molecules has grown into an impressive activity. A rich collection of structures has been identified, and many new amphiphilic molecules have been discovered or synthesized such as, for example, amphiphilic diblock copolymers. It is therefore no surprise that the very notion of micellization has become intimately connected to the amphiphilic nature of molecules. However, there is also another way to look at micellization, namely in terms of phase separation. A micelle can be regarded as a droplet of a new phase that has been arrested in its growth because part of the constituent molecules do not participate in the phase separation but remain in contact with the solvent.3 If one takes this perspective, any kind of phase separation could conceivably be the basis of colloidal particles spontaneously formed by selfassembly. If such an assembly has a size commensurate with molecular length scales, so that the participating molecules can span the entire object, one can denote these as micelles. In this paper we discuss the formation of a special kind of micelles which are not formed by a single, amphiphilic compound but are based on mixing polyelectrolytes of opposite charge. It is well-known that mixtures of polycations and polyanions can phase separate whenever the mixture is sufficiently close to isoelectric conditions.4-8 In early work, this type of phase separation has been called complex (1) Gelbart, W. M., Ben-Shaul, A., Roux, D., Eds. Micelles, Membranes, Microemulsions and Monolayers; Springer: New York, 1994. (2) McBain, J. W. Trans. Faraday Soc. 1913, 9, 99. (3) See, for example: Evans, D. F.; Wennerstro¨m, H. The Colloidal Domain; VCH: New York, 1994. (4) Bungenberg de Jong, H. G.; Kruyt, H. R. Proc. Acad. Sci. Amsterdam 1929, 32, 849. (5) Bungenberg de Jong, H. G. In Colloid Science; Kruyt, H. R., Ed.; Elsevier Publishing Company: Amsterdam, 1949; Vol. II, Chapters VIII and X. (6) Tsuchida, E.; Abe, K. Adv. Polym. Sci. 1982, 45, 2. (7) Zezin, A. B.; Kabanov, V. A. Russ. Chem. Rev. (Engl. Transl.) 1982, 51, 833. (8) See also, for example: Petrak, K. In Polyelectrolytes, Science and Technology; Hara, M., Ed.; Marcel Dekker: New York 1992; p 265. Dautzenberg, H.; Jaeger, W.; Ko¨tz, J.; Philipp, B.; Seidel, Ch.; Stscherbina, D. Polyelectrolytes, Formation, Characterization, and Application; Hanser Publishers: Munich, 1994; Chapter 6.
flocculation or complex coacervation,4,5 depending on whether the new phase is solid-like or liquid-like. Later, the terms polyelectrolyte complex (PEC) or interpolyelectrolyte complex (IPEC) have been used as well.6-8 The driving force for this type of phase separation is the entropy gain connected with the liberation of small counterions that were initially confined by the electric field of the participating polyions; the phase separation can therefore be suppressed by high salt concentrations. This is the basic idea behind the theoretical approach by Voorn.9-12 In the present study we achieve the arrested phase growth by means of a nonionic, water-soluble polymer chain which is end-to-end-attached to one of the polyions. This would lead to micelles consisting of an (approximately) isoelectric mixture of an A-B diblock copolymer (with one charged block and one hydrophilic neutral block) and a polyelectrolyte of opposite charge. Hence, none of the constituent molecules have amphiphilic properties with respect to the solvent (water). It is only upon mixing that a driving force for micellization arises. Micelles of this kind have been described previously by a few other investigators: Kabanov et al.,13 (who used the term “block ionomer complexes” (BICs), and Kataoka and co-workers,14-17 who prefer the term “polyion complex micelles” (PIC micelles). As for commonly known polymeric micelles, one anticipates the (relative) lengths of the chains forming the complex coacervate to play a role, but also the ionic strength must be important.9-12 We therefore studied the formation of micelles from mixtures of a polyacid (PAA) and a block copolymer with a cationic block. When we prepared the samples, we noted that there appeared to be a transient turbidity, as if a macroscopic phase formed initially. (9) Voorn, M. J. Ph.D. Thesis, Utrecht University, 1956. (10) Overbeek, J. Th. G.; Voorn, M. J. J. Cell. Comp. Physiol. 1957, 49, 7-26. (11) Voorn, M. J. Fortschr. Hochpolyn.-Forsch. 1959, 1, 192. (12) Voorn, M. J. Recl. Trav. Chim. 1956, 75, 317. (13) Kabanov, A. V.; Bronich, T. K.; Kabanov, V. A.; Yu, K.; Eisenberg, A. Macromolecules 1996, 29, 6797. (14) Harada, A.; Kataoka, K. Macromolecules 1998, 31, 288. (15) Kataoka, K.; Togawa, H.; Harada, A.; Yasugi, K.; Matsumoto, T.; Katayose, S. Macromolecules 1996, 29, 8556. (16) Harada, A.; Kataoka, K. Macromolecules 1995, 28, 5294. (17) Cammas, S.; Kataoka, K. In Solvents and Self-organisation of Polymers; Webber, S. E., et al., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1996; pp 83-113.
10.1021/la980778m CCC: $15.00 © 1998 American Chemical Society Published on Web 11/04/1998
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Figure 1. Structural formulas for the polymers used in this study: (a) poly((dimethylamino)ethyl methacrylate)-co-poly(glyceryl methacrylate) (PDMAEMA-co-PGMA); (b) poly(acrylic acid) (PAA). Table 1. Polymer Characteristics sample
no. of ionic monomers
no. of neutral monomers
molar mass (g/mol)
1:1 diblock 1:3 diblock 1:9 diblock poly(acrylic acid)
63 35 12 158
64 105 118
20 000 22 000 20 500 11 300
Experimental Section As the A-B diblock copolymer we used poly((dimethyl amino)ethyl methacrylate)-co-poly(glyceryl methacrylate) (PDMAEMAco-PGMA). The structure of this polymer is given in Figure 1. Both the A and the B block are entirely water soluble, the (dimethyl aminoethyl)methacrylate block being positively charged at pH < 9. The synthesis of this polymer by anionic polymerization and its characterization have been described elesewhere.18 Three samples differing essentially in the A/B monomer ratio were available; these are listed in Table 1. As the negatively charged polyelectrolyte we used poly(acrylic acid) (see also Figure 1) with a molar mass of 11 300. Solutions of PAA and block copolymer were simply mixed in various ratios around the composition which is stoichiometric with respect to charges. The solutions thus obtained were characterized by means of static and dynamic light scattering using an ALV light-scattering apparatus with an argon ion laser as the light source. The wavelength used was 514 nm, and all data were taken at the scattering angle 90°. The static intensity was obtained by averaging the photon count rate over periods of 10 s. The intensity fluctuations were analyzed automatically and in a single run by means of an ALV-2000 digital correlator over the entire range of correlation times from nanoseconds to milliseconds. For most samples, at least 20 runs were taken and the results were averaged after discarding those runs that had been spoilt by an occasional dust particle. The stopped-flow measurements were done in a Hi-Tech Scientific SHU SF-51 stopped-flow apparatus, at the fixed total polymer concentration 2500 mg/L of the 1:3 copolymer and 390 mg/L of PAA (this corresponds to about 5.6 mM acrylic acid monomers and 4.0 mM DMAEMA monomers). The scattered intensity (arbitrary units) under 90° and at the wavelength 514.5 nm was measured as a function of time. Cryo-TEM pictures were taken at the same concentration as that for the stopped-flow experiments. A copper TEM grid was immersed in the mixed polymer solution and blotted off, after which it was rapidly vitrified by immersion into liquid ethane near its freezing point. The blotting and freezing operations were carried out by an automat in order to ensure the best reproducibility. The vitrified sample was kept at boiling nitrogen temperature and transferred to a special, cooled sample stage of the electron microscope.
Results and Discussion In Figure 2 we present light-scattering results of various mixtures, plotted as a function of the composition given here as the ratio of chargeable groups. The pH was kept
Figure 2. Scattered intensity of a mixture of poly(acrylic acid) and the block copolymer at pH 7 as a function of the mixing ratio, expressed in the relative amounts of anionic and cationic chargeable groups. Vertical lines: block ratio 1:1; limits of unstable region. Solid curve: block ratio 1:3; forms micelles.
at 7 by making up all samples in a buffer with the approximate strength 0.8 mM. At this pH both polyions are partially charged (the cationic polymer somewhat more than the poly(acrylic acid)), such that the mixture is approximately isoelectric around the anionic/cationic stoichiometric ratio about 1.4. The concentration of block copolymer was kept fixed at 100 mg/L (corresponding to 0.16 mM cationic monomers), and the PAA concentration was varied between zero and the maximum concentration 30 mg/L (corresponding to 0.42 mM acrylic acid monomer units). The two vertical lines in the diagram refer to a block copolymer with a 1:1 block ratio (monomer composition) between the charged and the neutral block. For this copolymer, when mixed with poly(acrylic acid), very strong scattering is found between the two vertical lines. Clearly, this is the “complex flocculation” to be expected around the isoelectric composition (equal amounts of positive and negative charge). The scattering is so strong in this range (it can be detected even by the naked eye) that it largely exceeds the intensity range in Figure 1, so that we decided to represent it merely by the vertical lines. After some time, a sediment settles on the bottom of the sample tube. We conclude that this mixture is undergoing macroscopic phase separation. Apparently, the length of the glyceryl metacrylate block is insufficient to stop the growth of the complex coacervate droplets. Outside the region indicated by the vertical lines, the scattering remains low, indicating the absence of a macroscopic phase separation. However, this does by no means exclude the possibility that soluble, nonstoichiometric complexes are formed, as pointed out by Zezin et al.7 The second curve refers to a copolymer with a 1:3 block ratio; that is, the neutral block is three times longer than the charged one. This curve has a pronounced maximum at the isoelectric composition, but the scattering remains finite and stable over prolonged periods of time. To the naked eye, the sample is clear; no macroscopic phase separation and no sediment can be seen. The dynamic light-scattering experiments give no indication that particles of insoluble material occur in the system. Cryo-TEM pictures taken from a more concentrated mixture of PAA and 1:3 block copolymer, with the same composition as that corresponding to the maximum in Figure 2, are given in Figure 3. Large droplets, which might indicate macroscopic phase separation, were not (18) Hoogeveen, N. G.; Cohen Stuart, M. A.; Fleer, G. J.; Frank, W.; Arnold, M. Macromol. Chem. Phys. 1996, 197, 2553-2564.
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Figure 3. Cryo-TEM micrograph of the isoelectric mixture of poly(acrylic acid) and poly((dimethylamino)ethyl methacrylate)co-poly(glyceryl methacrylate). Small dark dots: micelles. Large dark circles: hexagonal ice formed during vitrification of the sample. Magnification: 73500×. Scale: 8 mm ) 200 nm.
found. Instead, there are many small dark dots in these pictures which are likely to be micelles. The size, as estimated from the pictures, is about 10 nm. Using reasonable values of the core density, one estimates from the size an aggregation number of order 10. Finally, we prepared mixtures of poly(acrylic acid) and a 1:9 diblock copolymer. Now, the scattering remains very low. Apparently, no colloidal objects (micelles) are present. Probably, the length of the polycation is too small to drive incipient phase separation; whether stable but soluble complexes are formed in this case cannot be concluded from the light-scattering data. As suggested by literature data,6 12 units are enough for a complex to form, but the minimum chain length required for stable complexes must also depend on ionic strength, and our data were obtained in the presence of some salt (0.8 mM). Also, micellization would bring several PGMA blocks together, which is unfavorable because the blocks, being in a good solvent, repel each other. We conclude from these observations that upon mixing with the appropriate amount of poly(acrylic acid) the 1:3 diblock system forms colloidal objects which do not grow toward a stable macroscopic phase. This is exactly the effect we expected the neutral, hydrophilic PGMA blocks to have: they are neutral and, hence, cannot be taken up in the insoluble complex, so that in order to remain solvated, they are forced to form a stabilizing corona, just like ordinary polymeric micelles.19 We therefore tentatively decide that we have a micellar system. Since the mechanism of phase separation underlying this micellar system is complex coacervation, that is, the association of oppositely charged polymers, one expects that addition of salt should suppress the micellization. In Figure 4 we present static light-scattering data for the micelle-forming system described above, studied at various salt concentrations. Clearly, the total scattered intensity drops rather sharply around 0.5 M NaCl, indicating that the micelles disappear. Kabanov et al.13 found a sharp drop around 0.35 M which is quite comparable. We should realize, however, that the stability of the complex will depend not only on the salt concentration but also on the hydrophobicity of the complexing polymers and the nature of the charged groups. We also studied the samples by dynamic light scattering (data not shown). The autocorrelation functions found were not singly exponential, indicating that the system (19) McConnell, G. A.; Gast, A. P.; Huang, J. S.; Smith, S. D. Phys. Rev. Lett. 1993, 71, 2102.
Cohen Stuart et al.
Figure 4. Scattered intensity for the isoelectric mixture of Figure 2 as a function of NaCl concentration.
Figure 5. Cryo-TEM micrograph of the same mixture as in Figure 2, showing the large “vesicle-with-enclosed-micelles” structures about 100 nm in size. These are possibly remnants of the macroscopic phase separation that precedes micelle formation. Scale: 12 mm ) 100 nm.
contained objects of different sizes. A fit to a sum of two exponentials gave a reasonable description. At low salt concentrations there is one fraction of very small objects (6-8 nm) which may well correspond to the dots observed in the cryo-TEM pictures. Another minor fraction at 6070 nm is also present. This fraction is likely to correspond with the large objects filled with black dots that are seen in some of the cryo-TEM micrographs; one example is shown in Figure 5. We regard these vesicle-like objects filled with micelles as incompletely equilibrated structures; they may be remnants of a macroscopic phase separation, as discussed below. At the salt concentrations 0.1 and 0.3 M, these large particles begin to swell, and at 1.0 M they have totally disappeared. The small objects also become somewhat larger, up to 10 nm, but at 1.0 M they have disappeared too. Figures 2-5 refer to fairly stable solutions as obtained at times larger than about 10 min after mixing the two starting polymer solutions. As pointed out above, similar observations were made by other investigators dealing with similar mixtures. A surprising and novel observation was the occurrence of transient phenomena. Immediately upon mixing, a strong scattering was initially observed, which subsequently disappeared. In Figure 6, we plot the transient excess scattering (with respect to a long-time baseline) versus time (logarithmic axis) as measured for the isoelectric mixture in a stopped-flow experiment under a scattering angle of 90°. Different salt concentrations were used, as indicated in the figure. All curves show a very rapid rise (the time resolution is not sufficient to see this) followed by a decay. The decay rate becomes progressively
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the initial polymer-rich domains, which have not had enough time to reorganize. The fact that no salt was added in these experiments and that the time between mixing and vitrification was a few minutes seems consistent with this idea.
Figure 6. Excess scattering of mixtures of poly(acrylic acid) and poly((dimethylamino)ethyl methacrylate)-co-poly(glyceryl methacrylate) as a function of time, in buffered solutions at pH 7 and for various concentrations of added NaCl, as indicated. In one experiment, buffer and salt were omitted. The strong scattering, indicating large fluctuations as normally found for macroscopic phase separation, dies away at a rate depending very strongly on salt concentration.
faster as the salt concentration increases, and finally, at 0.3 M NaCl, the transient turbidity disappears altogether. If only micelles would form as they are found in the stable solutions, the scattering would never show such nonmonotonic behavior. Hence, we conclude that, immediately after mixing, the system first phase separates on large length scales, after which the newly formed dense polymeric phase droplets reorganize and disintegrate into small structures. In our opinion, this is further proof that the system has a tendency to phase separate but that due to the presence of an uncharged block of appropriate length a macroscopic phase separation is unstable with respect to micellar aggregates. The higher the salt concentration, the faster the reoganization process runs. Qualitatively, this is what one expects, but the span of factor 104 between the extremes is remarkably large. We should note, however, that similar dramatic effects of added salt were found in studies of exchange processes occurring between polyelectrolyte complexes and added free polyions.20 It seems plausible that the peculiar objects observed in some cryo-TEM pictures (see Figure 4) are remnants of (20) Bakeev, K. N.; Izumrudov, V. A.; Kuchanov, S. I.; Zezin, A. B.; Kabanov, V. A. Macromolecules 1992, 25, 4249.
Conclusions We have studied the conditions under which small colloidal particles form as a result of complex coacervation. In dilute buffer solutions at pH 7, we find such particles when PAA of molar mass 11 300 is added to a solution of poly((dimethylamino)ethyl methacrylate)-co-poly(glyceryl methacrylate) (PDMAEMA-PGMA) with the block length ratio 1:3. The PAA block forms a complex (coacervate) with the PDMAEMA block, and the neutral hydrophilic PGMA block stabilizes small colloidal droplets of this coacervate. Since these particles are too small to have a proper bulk phase (i.e., consisting of molecules that do not interact with the interface), they may be called micelles. Upon mixing the two components, a micellar solution does not form initially. Instead, the mixture first undergoes macroscopic phase separation into a polymer-rich phase and a dilute phase; the former then spontaneously rearranges into a stable (micellar) solution. Such rearrangement processes are not known for conventional micellar systems. Added electrolyte strongly enhances the rate at which the rearrangement occurs: the rate increases by the factor 104 from pure water to 0.3 M NaCl. At salt concentrations beyond 0.5 M NaCl, micelles are no longer stable and disappear. Self-assembling systems based on polyelectrolyte complexes, like the one described above, may open up an entire new class of self-assembly with, as yet, unknown static and dynamic properties. Polymer chain length, polymer structure, pH, counterion type, and so forth are probably all important variables to be investigated in this context. We also anticipate very unusual adsorption behavior on charged surfaces. Acknowledgment. We would like to thank P. Bomans and P. M. Frederik (State University of Limburg, Maastricht) for taking the cryo-TEM pictures. We would also like to thank one of the reviewers for bringing some important references to our attention. LA980778M
