Assemblies of Double Hydrophilic Block Copolymers and Oppositely

Jan 5, 2009 - Frank Reinhold,† Ute Kolb,‡ Ingo Lieberwirth,† and Franziska Gröhn*,†,‡. Max Planck Institute for Polymer Research, Ackermann...
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Assemblies of Double Hydrophilic Block Copolymers and Oppositely Charged Dendrimers Frank Reinhold,† Ute Kolb,‡ Ingo Lieberwirth,† and Franziska Gro¨hn*,†,‡ Max Planck Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany, and Institut fu¨r Physikalische Chemie, UniVersita¨t Mainz, Welder-Weg 11, D-55099 Mainz, Germany ReceiVed August 24, 2008. ReVised Manuscript ReceiVed NoVember 11, 2008 The association of poly(ethylene oxide-b-methacrylic acid) and poly(amidoamine) dendrimers was examined by dynamic light scattering and small angle neutron scattering. With increasing amounts of the G4 dendrimer as the counterion, the size of the assemblies increases until it reaches a hydrodynamic radius of about 70 nm. The structure is consistent with poly(methyl methacrylate) (PMAA) chains closely aggregating with the dendrimers at low dendrimer amounts and volume-filling PMAA blocks at higher dendrimer contents. Similar behavior was observed for G4 and G2 dendrimers, while smaller G0 molecules showed an opposite dependence. The results represent an example of finite size assemblies formed by “electrostatic self-assembly” that are stable in aqueous solution and represent equilibrium structures, the structure and size of which can be tuned through the building units, loading ratio, and pH.

Introduction Ionic interaction between macroions and oppositely charged molecules plays a crucial role in the formation of structures in nature such as, for example, DNA-histone complexes. Therefore, also synthetic approaches for a structural design based on polyelectrolytes have become of interest.1-28 Combination of * To whom correspondence should be addressed. Fax: 49-6131-379100. E-mail: [email protected]. † Max Planck Institute for Polymer Research. ‡ Universita¨t Mainz. (1) Antonietti, M.; Conrad, J.; Thu¨nemann, A. Macromolecules 1994, 27, 6007–6011. (2) Antonietti, M.; Burger, C.; Thu¨nemann, A. Trends Polym. Sci. 1997, 5, 262–267. (3) Ra¨dler, J. O.; Koltover, I.; Salditt, T.; Safinya, C. R. Science 1997, 275, 810–817. (4) Koltover, I.; Salditt, T.; Ra¨dler, J. O.; Safinya, C. R. Science 1998, 281, 78–81. (5) Ra¨dler, J. O.; Koltover, I.; Jamieson, A. Langmuir 1998, 14, 4272–4283. (6) Thu¨nemann, A. F. Langmuir 2000, 16, 824–828. (7) Zhou, S. Q.; Chu, B. AdV. Mater. 2000, 12, 545–556. (8) Tiitu, A.; Laine, J.; Serimaa, R.; Ikkala, O. J. Colloid Interface Sci. 2006, 301, 92–97. (9) Hansson, P.; Almgren, M.; Mukhtar, E.; Van Stamm, J. Langmuir 1992, 8, 2405–2412. (10) Hansson, P.; Almgren, M. Langmuir 1994, 10, 2115–2124. (11) Hansson, P.; Almgren, M. J. Phys. Chem. 1995, 99, 16684–16693. (12) Hansson, P.; Almgren, M. J. Phys. Chem. 1995, 99, 16694–16703. (13) Hansson, P.; Almgren, M. J. Phys. Chem. 1996, 100, 9038–9046. (14) Hayakawa, K.; Santerre, J. P.; Kwak, J. C. T. Macromolecules 1983, 16, 1642–1645. (15) Liu, J.; Takisawa, N.; Shirahama, K. J. Phys. Chem. B 1997, 101, 7520– 7523. (16) Malovikova, A.; Hayakawa, K.; Kwak, J. C. T. J. Phys. Chem. 1984, 88, 1930–1933. (17) Ko¨tz, J.; Kosmella, S.; Beitz, T. Prog. Polym. Sci. 2001, 26, 1199–1232. (18) Huber, K. J. Phys. Chem. 1993, 97, 9825–9830. (19) Ikeda, Y.; Beer, M.; Schmidt, M.; Huber, K. Macromolecules 1998, 31, 728–733. (20) Peng, S. F.; Wu, C. Macromolecules 1999, 32, 585–589. (21) Schweins, R.; Huber, K. Eur. Phys. J. E 2001, 5, 117–126. (22) Lin, W.; Zhou, Y. S.; Zhao, Y.; Wu, C. Macromolecules 2002, 35, 7407– 7413. (23) Schweins, R.; Lindner, P.; Huber, K. Macromolecules 2003, 36, 9564– 9573. (24) Schweins, R.; Goerigk, G.; Huber, K. Eur. Phys. J. E 2006, 21, 99–110. (25) Goerigk, G.; Huber, K.; Schweins, R. J. Chem. Phys. B 2007, 127, 154908. (26) Thu¨nemann, A. F.; Mu¨ller, M.; Dautzenberg, H.; Joanny, J. F. O.; Lo¨wen, H. AdV. Polym. Sci. 2004, 166, 113–171. (27) Ou, Z.; Muthukumar, M. J. Chem. Phys. 2006, 124, 154902. (28) Sto¨rkle, D.; Duschner, S.; Heimann, N.; Maskos, M.; Schmidt, M. Macromolecules 2007, 40, 7998–8006.

polyelectrolytes with oppositely charged dendrimers has attracted attention as a model system for DNA-protein interaction and dendrimer-DNA complexes have been shown to be gene transfer agents.29-33 Polyelectrolytes and ionic surfactants form wellorganized solid materials.1-8 For various applications with biological, pharmaceutical, or environmental focus it is however desirable to build complexes in aqueous solution. In solution, polyelectrolyte-surfactant complexes with nonstoichiometric charge ratios showed sizes comparable to those of micelles without a polyelectrolyte.9-16 Polyelectrolyte assemblies formed predominantly driven by electrostatic interactions rather than hydrophobic interaction may be of special interest because they can be influenced through the ionic strength and in the case of weak polyelectrolytes through the pH. Two major types of ionic polyelectrolyte aggregates have been investigated in detail: Polyelectrolytes with multivalent inorganic salts show aggregation due to intermolecular bridging at high salt concentration and a decrease of the polymer size due to intramolecular bridging at low salt concentration, while the size and coordination capability of the counterion (e.g., whether Cu2+ or Ca2+ is applied) play an additional decisive role.17-25 Further, aggregation of two oppositely charged polyelectrolytes yields complexes that have been described as ladderlike for small molecular weight components and scrambled-egg-like for high molecular weight components.26-28 However, in both cases, polyelectrolytes with multivalent inorganic ions and interpolyelectrolyte complexes, possibilities to direct the structure are limited and the size distribution of the aggregates is usually broad. Therefore, it became of interest to apply components with a certain “architecture” to tune the structure of the assemblies. Complex formation of poly(diallyldimethylammonium chloride) with ionic dendrimers depended on the surface charge density of the den(29) Plank, C.; Mechtler, K., Jr.; Wagner, E. Hum. Gene Ther. 1996, 7, 1437– 1446. (30) Kukowska-Latallo, J. F.; Bielinska, A. U.; Johnson, J.; Spindler, R.; Tomalia, D. A.; Baker, J. R., Jr Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4897– 4902. (31) Chen, W.; Turro, N. J.; Tomalia, D. A. Langmuir 2000, 16, 15–19. (32) Guillot-Nieckowski, M.; Joester, D.; Sto¨hr, M.; Losson, M.; Adrian, M.; Wagner, B.; Kansy, M.; Heinzelmann, H.; Pugin, R.; Diederich, F.; Gallani, J. L. Langmuir 2007, 23, 737–746. (33) Shi, X.; Wang, S; Meshinchi, S.; Antwerp, M. E. V.; Bi, X.; Lee, I.; Baker, J. R., Jr Small 2007, 3, 1245–1252.

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drimers.34,35 Cluster formation of smaller complexes was discussed for the case of poly(L-glutamate) and poly(amidoamine) (PAMAM) dendrimers.36-40 However, as is also the case for the other polyelectrolyte systems mentioned, precipitation occurred at the charge stoichiometry. Recently we have shown that dendrimers or cylindrical polyelectrolyte brushes and multivalent organic counterions can form defined assemblies of various shapes.41-43 In these systems, finite-size assemblies are stable in aqueous solution due to charge stabilization. Double hydrophilic block copolymers (DHBCs) that consist of a charged and an uncharged hydrophilic block can be used to increase the “solubility” of inorganic crystals and polyelectrolyte complexes because the uncharged block provides compatibility with the solvent and keeps the aggregated structure in solution.44-51 With multivalent inorganic salts soluble complexes with DHBCs were formed, but the possibilities to control the shape and size of such aggregates are limited.52-54 With two oppositely charged DHBCs soluble products were formed in a small pH range at charge ratios around 1:1.55-59 In this study we investigate the combination of DHBCs with oppositely charged dendrimers. It is expected that DHBCs allow for the formation of complexes stable in aqueous solution over a larger parameter range than homopolyelectrolytes. Thereby, for fundamental questions a more systematic investigation as a model system is possible, and new types of aggregates for potential applications may arise in addition. Small dendrimers are applied as “counterions” because they are available with a range of radii and possess a well-defined number of chargeable groups.60-63 Due to the smaller number of charges and limited degrees of conformational freedom as compared to those of a linear flexible polyelectrolyte, dendrimer-polyelectrolyte complexes may result (34) Zhang, H.; Dubin, P. L.; Spindler, R.; Tomalia, D. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 923–928. (35) Zhang, H.; Dubin, P. L.; Ray, J.; Manning, G. S.; Moorefield, C. N.; Newkome, G. R. J. Phys. Chem. B 1999, 103, 2347–2354. (36) Leisner, D.; Imae, T. J. Phys. Chem. B 2003, 107, 8078–8087. (37) Imae, T.; Mitra, A. J. Phys. Chem. B 2003, 107, 80888092.u. (38) Leisner, D.; Imae, T. J. Phys. Chem. B 2003, 107, 13158–13167. (39) Leisner, D.; Imae, T. J. Phys. Chem. B 2004, 108, 1798–1804. (40) Mitra, A.; Imae, T. Biomacromolecules 2004, 5, 69–73. (41) Gro¨hn, F.; Klein, K.; Brand, S. Chem.sEur. J. 2008, 14, 6866–6869. (42) Willerich, I.; Gro¨hn, F. Chem.sEur. J. 2008, 14, 9112–9116. (43) Gro¨hn, F. Macromol. Chem. Phys. 2008, 209, 2295–2301. (44) Co¨lfen, H. Macromol. Rapid Commun. 2001, 22, 219–252. (45) Thu¨nemann, A. F.; Beyermann, J.; Kukula, H. Macromolecules 2000, 33, 5906–5911. (46) Faatz, M.; Gro¨hn, F.; Wegner, G. Mater. Sci. Eng., C 2005, 25, 153–159. (47) Gorna, K.; Munoz-Espi, R.; Gro¨hn, F.; Wegner, G. Macromol. Biosci. 2007, 7, 163–173. (48) Co¨lfen, H. Biomineralization II 2007, 271, 1–77. (49) Giacomelli, C.; Schmidt, V.; Borsali, R. Macromolecules 2007, 40, 2148– 2157. (50) Xu, A. W.; Ma, Y. R.; Co¨lfen, H. J. Mater. Chem. 2007, 17, 415–449. (51) Cheng, C.; Wei, H.; Shi, B. X.; Cheng, H.; Li, C.; Gu, Z. W.; Cheng, S. X.; Zhang, X. Z.; Zhuo, R. X. Biomaterials 2008, 29, 487–505. (52) Li, Y.; Gong, Y. K.; Nakashima, K. Langmuir 2002, 18, 6727–6729. (53) Sanson, N.; Bouyer, F.; Ge´rardin, C.; In, M. Phys. Chem. Chem. Phys. 2004, 6, 1463–1466. (54) Franc¸ois, J.; Truong, N. D.; Medjahdi, G.; Mestdagh, M. M. Polymer 1997, 38, 6115–6127. (55) Gohy, J. F.; Varshney, S. K.; Jeroˆme, R. Macromolecules 2001, 34, 3361– 3366. (56) Holappa, S.; Andersson, T.; Kantonen, L.; Plattner, P.; Tenhu, H. Polymer 2003, 44, 7907–7916. (57) Harada, A.; Kataoka, K. Macromolecules 1995, 28, 5294–5299. (58) Harada, A.; Kataoka, K. Science 1999, 283, 65–67. (59) Andersson, T.; Holappa, S.; Aseyev, V.; Tenhu, H. J. Polym. Sci., Part A: Polym. Chem. 2003, 41, 1904–1914. (60) Prosa, T. J.; Bauer, B. J.; Amis, E. J.; Tomalia, D. A.; Scherrenberg, R. J. Polym. Sci., Part B: Polym. Phys. 1997, 35, 2913–2924. (61) Bosman, A. W.; Janssen, H. M.; Meijer, E. W. Chem. ReV. 1999, 99, 1665–1688. (62) Gro¨hn, F.; Bauer, B. J.; Akpalu, Y. A.; Jackson, C. L.; Amis, E. J. Macromolecules 2000, 33, 6042–6050. (63) Cakara, D.; Kleimann, J.; Borkovec, M. Macromolecules 2003, 36, 4201– 4207.

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in more defined structures than interpolyelectrolyte complexes. In addition, such aggregates could open new possibilities by combining the capability of dendrimers to act as a host with the special properties of a supramolecular aggregate.62,64,65 Mitra et al. used a cationic copolymer with dendrimer counterions but found no large differences in homo- and copolymer complexation and precipitation.66 Our goal herein is to investigate whether the double hydrophilic poly(methacrylic acid-block-ethylene oxide) (PMAA-b-PEO) and PAMAM dendrimers can form watersoluble complexes and to analyze the shape and size of the structures in solution.

Experiments Materials. Poly(amidoamine) dendrimers were purchased from Aldrich. Sodium hydroxide (1 N) and sodium deuteroxide (30 wt %) were obtained from Aldrich and potassium dihydrogen phosphate (>99%) and deuterium oxide (99.9%) from Merck and Deutero GmbH, respectively. Poly(methacrylic acid-b-ethylene oxide) was obtained from Polymersource, Inc., Quebec, Canada. Characterization was performed by size exclusion chromatography (SEC) and by titration with sodium hydroxide. The block lengths of the copolymer are N(EO) ) 170 and N(MAA) ) 203. Sample Preparation. The pH 6 and pH 7 buffer solutions were prepared as explained elsewhere.67 Samples were prepared from stock solutions of the copolymer and the dendrimer. The polymer concentration cp was fixed, and the concentration of the counterion (dendrimer) was varied to realize a specific loading ratio. Light Scattering. Sample solutions were filtered three times with Millipore Millex-HA 0.45 µm filters before the cells were filled with them. Measurements were carried out with an ALV-5000 photometer of ALV-Laser Vertriebs-GmbH, Langen, Germany, using a krypton ion laser (647.1 nm) or a Nd:YAG laser (532 nm). Single cross correlation of two avalanche photodiodes was used at angles ranging from 30° to 150° (in 15° steps). Data analysis was performed by inverse Laplace transformation under regularization using CONTIN, and the obtained diffusion coefficients were extrapolated to zero scattering vector. Hydrodynamic radii result from the Stokes-Einstein relationship. Error bars of the hydrodynamic radii as obtained from the linear regression of the angle-dependent measurements are within the symbol size. Small Angle Neutron Scattering. Buffer solutions were prepared with deuterium oxide, potassium dihydrogen phosphate, and sodium deuteroxide. The measurements were carried out at the Forschungszentrum Ju¨lich. The wavelength of 7 Å was adjusted with a velocity selector (∆λ/λ ) 0.2), and a 60 × 60 cm2 6Li glass detector was used. The samples were measured at sample-detector distances of 1.25, 2, 8, and 20 m covering a range of 0.025 nm-1 < q < 2.5 nm-1. The calibration was performed with tetraborcarbide, and the measured values were corrected by dark counts (measured with Cd), detector sensibility (measured with plexiglass) and background scattering of the cell and solvent. Experimental errors of I (q) data are within 1% for q < 0.5 nm-1, are below 10% for q < 1 nm-1 and increase to 100% for highest q data. Cryo Transmission Electron Microscopy (Cryo-TEM). The sample was vitrified on Quantifoil (2 µm hole size) from aqueous solution in liquid propane using an FEI Vitrobot at room temperature. TEM measurements were performed using a Gatan cryotransfer sample holder in an FEI Tecnai12 with a LaB6 cathode at 120 kV acceleration voltage.

Results and Discussion When PEO-b-PMAA and G4 PAMAM dendrimer were combined, opalescence of the resulting samples already indicated (64) Jansen, J.F.G.A; de Brabander-van den Berg, E. M. M.; Mejier, E. W. Science 1994, 266, 1226–1229. (65) Jansen, J.F.G.A; Mejier, E. W. J. Am. Chem. Soc. 1995, 117, 4417–441. (66) Mitra, N.; Dubin, P. L.; Moorefield, C. N.; Newkome, G. R. Langmuir 1999, 15, 4245–4250. (67) Handbook of Chemistry and Physics, 76th ed.; Lide, D. R., Ed.; CRC Press: Boca Raton, FL, 1995- 1996.

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Figure 1. Dynamic light scattering on PEO-b-PMAA/G4 samples with different loading ratios l at pH 7: electric field autocorrelation function g1(τ) (O) and distribution of relaxation times ΓA(τ) (b); (a) l ) 0.25; (b) l ) 2; cp ) 0.5 g L-1 for both panels.

the formation of larger aggregates in aqueous solution. The association depended on the polymer concentration cp and mixing ratio of the components. With increasing dendrimer concentration at constant polymer concentration the samples became more opaque, but no precipitation was observed. In the following the ratio of the molar concentration of primary dendrimer amine groups to the molar concentration of methacrylic acid units of the copolymer is defined as the loading ratio l

l)

c(-NH2, dendrimer) c(-COOH, copolymer)

(1)

in analogy to the inverse parameter λ used for the description of polyelectrolytes by Fo¨rster et al.68-72 In pH 7 buffer solution opalescence is observed for samples with l g 0.3. In pH 6 buffer solution opalescence is observed already at l g 0.1. Turbidity measurements show no changes in a temperature range between 5 and 50 °C. The aggregation was further monitored by dynamic light scattering (DLS). For higher loading ratios (l g 0.5) the scattering intensitywasfoundtobe10-foldhigherforthecopolymer-dendrimer solutions than for the copolymer or the dendrimer solution separately, clearly indicating aggregate formation. For l ) 0.25 at pH 7 coexistence of just a small amount of aggregates next to small species, likely copolymer chains, was observed (Figure 1a). For all samples with larger loading ratios only one welldefined diffusion process was measured by dynamic light scattering, as shown in Figure 1b. As the ionic strength of the buffer solutions is large enough to screen the charges of the polyelectrolyte (Is ≈ 0.07 M), no “special polyelectrolyte diffusion behavior” in terms of two diffusive processes is expected, and the measured diffusion coefficient can be translated into a hydrodynamic radius of species in solution. When aggregated particles formed from smaller building units are observed, it is of interest whether their formation is kinetically controlled (as is usually the case for interpolyelectrolyte complexes26-28) or they represent equilibrium structures (as we found for assemblies from smaller building units41-43). Most easily, this can be tested by preparing samples with the same final compositions via different preparation routes. Figure 2 shows dynamic light scattering results in the form of autocorrelation functions and distribution of relaxation times for samples with a polymer concentration cP ) 1 g L-1 and loading ratio l ) 1 (68) Fo¨rster, S.; Schmidt, M.; Antonietti, M. Polymer 1990, 31, 781–792. (69) Sedlak, M.; Amis, E. J. J. Chem. Phys. 1992, 96, 826–834. (70) Gro¨hn, F.; Topp, A.; Belkoura, L.; Woermann, D. Phys. Chem. Chem. Phys. 1995, 99, 736–740. (71) Ermi, B. D.; Amis, E. J. Macromolecules 1998, 31, 7378–7384. (72) Antonietti, M.; Briel, A.; Gro¨hn, F. Macromolecules 2000, 33, 5950– 5953.

Figure 2. Dynamic light scattering on PEO-b-PMAA/G4 samples (l ) 1, cp ) 1 g L-1, at pH 6) obtained via different preparation routes: black circles, addition of G4 to copolymer under stirring; red circles, addition of copolymer to G4 under stirring; green circles, addition of G4 to copolymer at once, followed by stirring; blue circles, addition of copolymer to G4 at once, followed by stirring.

at pH 6 resulting from different mixing orders and extend of stirring. It is evident that independently of whether the copolymer was added to the dendrimer solution or vice versa, aggregates with an average hydrodynamic radius of about RH ) 50 nm (46-54 nm) were observed. This size was also reproducible when a sample was prepared several times the same way. The distribution width varies between 18% and 22%. Parts a and b of Figure 3 show the results at cP ) 0.5 and 1 g L-1 in pH 7 and 6 buffer solution dependent on the loading ratio l. Upon increasing addition of dendrimers to copolymer, that is, with increasing l, the hydrodynamic radii increase to RH ) 60-70 nm. At pH 7 the complexation starts at 0.25 < l < 0.5 (4-fold excess of chargeable groups of the polymer), whereas at pH 6 even at l ) 0.1 (about 8-fold excess of chargeable groups of the polymer) particles larger than the copolymer are detected. For l > 1 the size of the complexes remains approximately constant in pH 7 buffer solution with hydrodynamic radii RH ) 60-70 nm. The same can be observed at pH 6, but there is a slight maximum in size at l ) 0.5. While the general dependence on the loading ratio is the same for different absolute concentrations, in pH 7 buffer solution slightly larger radii are observed for cp ) 1 g L-1 as compared to cp ) 0.5 g L-1. In contrast, in pH 6 buffer solution, a decrease of the hydrodynamic radius is observed with increasing polymer concentration from 0.25 to 2 g L-1 (shown are 0.5 and 1 g L-1). The formation of aggregated structures was confirmed by cryoTEM as shown in Figure 4. It can be observed that the complex particles to a first approximation have a somewhat “imperfect” sphere shape. Although no staining agent was applied, the contrast

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Figure 3. Hydrodynamic radius of samples consisting of PEO-b-PMAA and PAMAM dendrimer G4 as a function of the loading ratio l as determined from DLS: (a) pH 7; (b) pH 6; (b) cp ) 0.5 g L-1; (O) cp ) 1 g L-1. Lines are to guide the eyes.

of the aggregates is well-expressed. This is most likely due to buffer ion, potassium and phosphate, accumulation in the ionic aggregate. In regions with high particle concentration the distance between these contrast-rich species remarkably is constant. It is about 13 nm, which we attribute to PEO chains surrounding the aggregate particles. PEO coils at the surface of two adjacent particles each with a diameter of about 6.5 nm may determine this distance. In contrast to the inner particle in which ionic entities and PEO should be contained close to each other (see the discussion below), the uncharged PEO in the shell may attract phosphate ions less strongly and thus show no expressed contrast in cryo-TEM. The size distribution resulting from Figure 4a is shown in Figure 4b. It yields an average diameter of 71 nm for the “dark” species. To compare this value with the dynamic light scattering results, the proposed PEO shell with less contrast must be added; that is, particles in TEM have an average total diameter of 84 nm. This is smaller than results from the hydrodynamic radius measured in DLS when solid spheres are assumed, which yields a diameter of 100 nm (RH ) 50 nm) for this sample. This may be due to the fact that larger particles scatter much more strongly and thus in the scattering experiment contribute to the average much more as in microscopy. In addition, the statistics of TEM is more for an impression rather than being representative at this point. Further, a cryo-TEM image of a more diluted sample is given in Figure 4c. It shows individual particles with smaller radii, which is in accordance with the concentration dependence observed in dynamic light scattering. Small angle neutron scattering (SANS) was performed to further investigate the structure of the aggregates. Figure 5a shows the scattering curves at different charge ratios in pH 7 buffer solution. One can clearly observe the differences in scattering intensity and scattering curve between aggregated particles (l ) 0.5, 1, and 2) and nonaggregated particles (copolymer and l ) 0.25). The radii of gyration, calculated by extrapolation of the linear Guinier plots (not shown), show a continuous increase starting at the radius of the copolymer. Pair distance distribution functions P(r) obtained from this measurement by indirect Fourier transformation of the I(q) data (Figure 5b) reveal information about the shape of the particles.73-75 The comparison with different models shows that they cannot be described by a sphere model, even a polydisperse one. A slightly ellipsoidal structure is in accordance with the data. The intersection of the function with the x-axis gives the maximum particle dimension (geo(73) Glatter, O. Acta Phys. Austriaca 1977, 47, 83–102. (74) Glatter, O. J. Appl. Crystallogr. 1977, 10, 415–421. (75) Moitzi, C.; Portnaya, I.; Glatter, O.; Ramon, O.; Danino, D. Langmuir 2008, 24, 3020–3029.

metrical diameter). It is D ≈ 110, 140, and 150 nm corresponding to radii R ≈ 55, 70, and 75 nm for l ) 0.5, 1, and 2, respectively. Thus, the same trend is observed as derived from dynamic light scattering. For l ) 2 some additional larger aggregates may be present in the solution. As mentioned above, the starting point of observing larger aggregates upon addition of the dendrimer to the copolymer depends on the pH: For pH 7 aggregates are first observed at l ) 0.5, while for pH 6 they are already observed at l ) 0.1. For further elucidation, the loading ratio l representing a molar ratio of chargeable groups can be translated into the charge ratio lcharge, representing the ratio of charged groups as derived from the degree of dissociation of both components at the respective pH resulting from titration curves. This yields lcharge ) 1.35 at l ) 0.5 for pH 7 and l charge ) 1.30 at l ) 0.1 for pH 6. Thus, the onset of aggregation is observed at similar charge ratios. Figure 6 shows the hydrodynamic radius as a function of the charge ratio. Aggregation starts at a range of 0.6 < lcharge < 1.35, that is, around the charge stoichiometry, and the hydrodynamic radius does not change much for lcharge > 5. Thus, the charge ratio is an important parameter in the aggregation, yet this is not the only influence as the hydrodynamic radius of the aggregates as a function of the charge ratio still shows a different dependence for pH 6 and 7, as seen in Figure 6. One reason for the choice of dendrimers in this model system was that different sizes (generations) can be compared. The copolymer was combined with PAMAM dendrimers of generations 0, 2, and 4 (G0, G2, and G4). The data are shown in Figure 7. Generation 2 shows the same trend as generation 4, that is, an increasing hydrodynamic radius with increasing charge ratio (open symbols). Contrarily the smallest dendrimer shows a different behavior: At l ) 0.1 (lcharge ) 1.15) no complexation occurs. It starts at l ) 0.25 (lcharge ) 2.3) with a hydrodynamic radius of RH ) 84 nm, which decreases with increasing counterion concentration to around RH ) 50 nm (full symbols). The results thus show that particles of up to RH ) 70 nm with slightly ellipsoidal shape are formed from poly(ethylene oxideb-methacrylic acid) and PAMAM dendrimers. The size of the PEO-b-PMAA dendrimer assemblies cannot be explained by a simple association model in analogy to amphiphilic copolymer micelles,76-81 such as the poly(methacrylic acid) blocks connected (76) Zhang, L. F.; Eisenberg, A. Science 1995, 268, 1728–1730. (77) Zhang, L. F.; Eisenberg, A. J. Am. Chem. Soc. 1996, 118, 3168–3181. (78) Fo¨rster, S.; Hermsdorf, N.; Bo¨ttcher, C.; Lindner, P. Macromolecules 2002, 35, 4096–4105. (79) Antonietti, M.; Fo¨rster, S. AdV. Mater. 2003, 15, 1323–1333. (80) Fo¨rster, S.; Abetz, V.; Mu¨ller, A. H. E. Polyelectrolytes Defined Mol Archit. II 2004, 166, 173–210.

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Figure 5. (a) SANS scattering curves I(q) of PEO-b-PMAA and PAMAM dendrimer G4 at cp ) 2 g L-1 and pH 7 at different charge ratios: (0) copolymer; (4) sample with l ) 0.25; (]) sample with l ) 0.5; (O) sample with l ) 1; (") sample with l ) 2. (b) Corresponding pair distance distribution functions P(r) for the larger complexes: symbols as in (a). I(q) and P(r) are in arbitrary units.

Figure 6. Hydrodynamic radius of PEO-b-MAA and the PAMAM G4 dendrimer at cp ) 0.5 g L-1 at pH 7 (O) and pH 6 (b) and different charge ratios as determined from DLS. Lines are to guide the eyes.

Figure 4. Cryo-TEM picture of PEO-b-PMAA/G4 samples in pH 7 buffer solution at l ) 0.5: (a) cp ) 0.5 g L-1; (b) size distribution according to (a); (c) cp ≈ 0.1 g L-1 (smaller particles at lower concentration).

by the dendrimers in the center and the poly(ethylene oxide) blocks as stabilizers outside. The end-to-end distance estimated for the PEO coil (5 nm) is substantially smaller and even the contour length of the PEO chain (64 nm) is somewhat smaller than the radius of the assemblies at low charge ratios. Thus, in the aggregate structures formed some of the poly(ethylene oxide) chains have to be in the inside of the aggregate too. (81) Azzam, T.; Eisenberg, A. Langmuir 2007, 23, 2126–2132.

From small angle neutron scattering one can calculate the apparent molar mass of the complexes. The scattering lengths of the PAMAM dendrimer and PEO-b-PMAA are similar, so this calculation is possible. The solvent-corrected intensity was extrapolated to q2 ) 0 in the Guinier regime at small q values. In the samples where aggregation of the copolymer and dendrimer takes place no unaggregated PEO-b-PMAA chains or dendrimers were observed by dynamic light scattering. Thus, we assume that the total amount of polymer and dendrimer is associated and the molar ratio of both components in the assembly is the same as in the total. As the ratio of dendrimer to copolymer and their molar masses are known, this also yields the number of dendrimers Ndend in the aggregate. This experimental number is shown as a function of the loading ratio as a solid line in Figure 8.

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Figure 7. Hydrodynamic radius of PEO-b-PMAA with PAMAM dendrimers of different generations as a function of the charge ratio (cp ) 0.5 g L-1, pH 6): (O) G4 dendrimer; (0) G2 dendrimer; (b) G0 dendrimer.

Figure 8. Number of dendrimers Ndend in complexes of PEO-b-MAA and PAMAM dendrimer G4 in pH 6 buffer solution at cp ) 2 g L-1 and different loading ratios λ as determined from SANS: (O) experimental values; (0) model 1; (∆) model 2.

This number can be compared with two different theoretical values that result from the experimentally determined aggregate volumes and an estimate of the volume of the components. The radius of gyration of the dendrimer is known from the literature,59 whereas the radius of gyration of the copolymer was obtained from SANS measurements. Model 1 assumes that the dendrimer keeps its swollen volume and the copolymer needs the same volume as in an unaggregated coil state in solution. In model 2 it is assumed that the poly(methacrylic acid) block of the copolymer does not fill any extra volume as it is closely attached

Reinhold et al.

to the dendrimer and thus a denser structure results. Both models are depicted in Figure 9. Figure 8 shows that at a large loading ratio (l ) 2) the number of dendrimers in the complex Ndend obtained from the experiments is similar to that which is calculated for model 1. At smaller loading ratios model 1 underestimates the number of dendrimers in the complex. That means there is a denser packing of the copolymer and the dendrimer counterions than expected from the sum of the volumes of both molecules. The poly(methacrylic acid) block may be attached to the dendrimer in such a way that the volume of this part of the macromolecule can be neglected for volume calculations (model 2). The number of molecules in the aggregated particle according to this model is consistent with the experimental value for l ) 0.25. The reason for these results may be that at low dendrimer content the poly(methacrylic acid) parts of the copolymer chains interact with the amino groups of the dendrimers and surround the dendrimers and connect them. Thus, they do not contribute much to the volume of the complex, and only the coiled PEO part and the dendrimer volume have to be taken into account. With increasing amount of dendrimer counterions, more dendrimers connect to the PMAA blocks. Thereby, the size of the aggregates increases, but the “connecting segments” between the two oppositely charged molecules become smaller. Free charges on the dendrimer surface additionally lead to a repulsion which forces the chain to occupy a larger volume to separate them. Accordingly, the size of the complexes remains constant when it is not possible any more to attach more dendrimers to a PMAA chain. The contour length of the PMAA block is 51 nm. That means that at maximum no more than 9.6 dendrimers (diameter 5.3 nm) geometrically fit next to each other on such a chain, to a first approximation (not taking into account repulsion of the free charges of the dendrimers, etc.). This corresponds to the number of dendrimers per chain at l ) 3 so that due to the spatial arrangement for l > 3 no further increase of the aggregate size is expected. With respect to the additional electrostatic repulsion, an even smaller value of l where the aggregate size stops increasing (experimentally for l > 2) can be understood. It should also be noted that a small amount of additional nonaggregated G4 dendrimers may not be detectable next to the larger aggregates by dynamic light scattering due to the much lower scattering inetnsity. Thus, it could be that at some point additional dendrimers are not incorporated into the aggregates anymore. The volume and the amount of solvent molecules in the complex are neglected in this simplified view as it is assumed that the degree of swelling of the components is the same in the

Figure 9. Cartoon of the proposed inner structure of complexes formed by PEO-b-PMAA and the G4 PAMAM dendrimer: (left, model 1) all entities (dendrimers, PEO, and PMAA) contribute to the aggregate volume (looser structure at large l); (right, model 2) the PMAA block is bound to the dendrimer surface and does not contribute to the volume of the aggregates (denser structures at small l).

DHBC and Oppositely Charged Dendrimer Assemblies

aggregated and unaggregated states. In addition, it is assumed that the complete volume is filled with (swollen) polymeric material, while an alternation of more densely aggregated moieties with looser connections and larger “holes” within the aggregate also represents a possible structure. Further, a much larger influence of the solvent on the structure of the aggregate is possible. In that respect, the suggested models represent the most simplified models for fundamental comparison and demonstration of structural changes with the loading ratio. In contrast, for the G0 dendrimer, a small molecule with four charges, addition of more counterions may lead to a closer connection of copolymers, that is, a decreasing aggregate size with increasing l. Therefore, we have shown that in addition to the charge ratio also the counterion size (and thereby charge) is of importance in the structure formation. In analogy to the systems described in refs 41-43 it is likely that aggregates of a finite size are stabilized due a combination of charge effects and geometric effects. A quantitative understanding as to what determines the absolute size of the aggregate and restricts its further growth is however still to be developed.

Conclusions We demonstrated the formation of defined supramolecular assemblies based on “electrostatic self-assembly” in aqueous solution. Specifically, it was shown that poly(ethylene oxideb-methacrylic acid) and PAMAM dendrimers associate into stable assemblies of varying size. While self-assembly based on amphiphilicity is a classical field, self-assembly based on ionic interaction had been investigated less intensively and in particular often had not led to narrowly distributed assemblies. In this system of ionic copolymer-dendrimer assemblies, with increasing amount of counterion G4, the size of the assemblies increases until it reaches a hydrodynamic radius of about 70 nm. The structure is consistent with poly(methyl methacrylate) (PMMA) chains closely aggregating with the dendrimers at low dendrimer amount and volume-filling PMMA blocks at higher dendrimer content. A similar behavior was observed with G4 and G2

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dendrimers, while the small G0 molecules showed an opposite influence of the loading ratio on the aggregate size. Thus, the size of the counterion and the counterion/copolymer ratio are important factors in determining the size and density of the assemblies. Most importantly, the results of this study demonstrate that assemblies of a defined size and “density” can be obtained by the association of ionic building blocks in solution. As compared to the usually kinetically controlled aggregation of two high molecular mass linear polyelectrolytes, the assemblies described in this study represent equilibrium structures and exhibit a relatively narrow size distribution. In addition, our previous hypothesis that the use of building units of a certain architecture allows for a better control of the structure and size as compared to flexible or very small (e.g., multivalent metal) ions is confirmed. Further, the combination of poly(ethylene oxide-b-methacrylic acid) with PAMAM dendrimers yielded assemblies stable in aqueous solution over a large parameter range. Therefore, the results presented establish electrostatic self-assembly as a versatile route for the formation of nanoassemblies with tunable size and structure in solution. In the future, further variation of the building blocks may lead to a larger structural variety of the assemblies formed. In addition, the capability of dendrimers to act as “hosts” for smaller guest molecules may be combined with the assembly formation described in this study, yielding more complex functional nanoscale assemblies. Acknowledgment. The scattering experiments were performed at KWS1, Ju¨lich Center for Neutron Science (JCNS), and D11, Institut Laue-Langevin (ILL), Grenoble, France. We thank Dr. Wim Pyckhout-Hintzen, JCNS, and Dr. Ralf Schweins, ILL, for help with the scattering experiments. We thank Katja Klein, Sabrina Brand, Christine Rosenauer, and Beate Mu¨ller for their contributions to this work and Prof. Dr. Manfred Schmidt and Prof. Dr. Gerhard Wegner for fruitful discussions. Financial support of the Max Planck Society, DFG (Grant SFB 625), and ILL is gratefully acknowledged. LA8027594