Dendrimer Influenced Supramolecular Structure Formation of Block

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Dendrimer Influenced Supramolecular Structure Formation of Block Copolymers: II. Dendrimer Concentration Dependence Xingfu Li, Anja Kroeger, Tony Azzam, and Adi Eisenberg* Department of Chemistry, McGill UniVersity, 801 Sherbrooke Street West, Montreal, Quebec, H3A 2K6, Canada ReceiVed August 23, 2007. In Final Form: NoVember 29, 2007 The dendrimer concentration dependence of the supramolecular structure formation of polystyrene-block-poly(acrylic acid) in dioxane/THF was investigated as a function of water content. The distribution as well as the localization of the dendrimer units inside the formed aggregates were determined by comparative studies of turbidity measurements and transmission electron microscopy. The strong and specific interactions present between the amine groups of the dendrimer (PAMAM) and the carboxylic acid residues of PAA in the copolymer have a strong influence on the structure formation. The PAMAM concentration as well as the character of the terminal groups of the dendrimer influence the strength of these interactions and consequently affect the structure formation process. As shown by fluorescence quenching experiments, on all supramolecular hierarchical structure levels, and specifically in vesicles, the dendrimer is coated by the PAA chains of the block copolymer due to the strong interactions; since the PAA blocks are connected to the PS blocks, which form the corona, the dendrimer is surrounded by PS chains and is thus encapsulated into the hydrophobic regions of the block copolymer aggregates. A high-resolution transmission electron microscopy image of a micelle is shown, in which the individual dendrimer cores are seen to be localized in the center of these aggregates, and thus, the structure proposed in the previous publication (Kroeger, A.; Li, X.; Eisenberg, A. Langmuir 2007, 23, 10732) is confirmed. Furthermore, the sizes of the resulting aggregates depend on the relative concentration of dendrimer, expressed as RAm/Ac (the ratio of amine to acid groups). With increasing RAm/Ac values, not only the sizes of the micelles but also the vesicle dimensions, especially vesicle wall thicknesses, increase, and this effect suggests the encapsulation of the dendrimer into the vesicle walls. Thus, the constitution of the vesicle structure is determined precisely. This feature allows the potential incorporation of a wide range of species into the vesicle walls or the center of the micelle cores.

Introduction The formation of nanostructured materials produced by molecular self-organization has attracted significant scientific attention. Amphiphilic block copolymers, which self-assemble in selective solvents or solvent mixtures into colloidal-sized aggregates of various morphologies, are ideal materials in this respect and have been studied intensively.1-20 The self-assembled * Corresponding author. E-mail: [email protected]. (1) van Hest, J. C. M.; Delnoye, D. A. P.; Baars, M. W. P. L.; Van Genderen, M. H. P.; Meijer, E. W. Science (Washington, DC, U.S.) 1995, 268, 1592. (2) Zhang, L.; Eisenberg, A. Science (Washington, DC, U.S.) 1995, 268, 1728. (3) Discher, D. E.; Eisenberg, A. Science (Washington, DC, U.S.) 2002, 297, 967. (4) Lee, J. C.-M.; Santore, M.; Bates, F. S.; Discher, D. E. Macromolecules 2002, 35, 323. (5) Antonietti, M.; Foerster, S. AdV. Mater. 2003, 15, 1323. (6) Zhang, L.; Yu, K.; Eisenberg, A. Science (Washington, DC, U.S.) 1996, 272, 1777. (7) Zhang, L.; Bartels, C.; Yu, Y.; Shen, H.; Eisenberg, A. Phys. ReV. Lett. 1997, 79, 5034. (8) Yu, Y.; Eisenberg, A. J. Am. Chem. Soc. 1997, 119, 8383. (9) Yu, G.-E.; Eisenberg, A. Macromolecules 1998, 31, 5546. (10) Kita-Tokarczyk, K.; Grumelard, J.; Haefele, T.; Meier, W. Polymer 2005, 46, 3540. (11) (a) Shen, H.; Eisenberg, A. Angew. Chem. 2000, 112, 3448. (b) Ibid, Angew. Chem., Int. Ed. 2000, 39, 3310. (12) Azzam, T.; Eisenberg, A. Angew. Chem., Int. Ed. 2006, 45, 7443. (13) Nardin, C.; Hirt, T.; Leukel, J.; Meier, W. Langmuir 2000, 16, 1035. (14) Ding, J.; Liu, G. Macromolecules 1997, 30, 655. (15) (a) Santore, M. M.; Discher, D. E.; Won, Y.-Y.; Bates, F. S.; Hammer, D. A. Langmuir 2002, 18, 7299. (b) Nukova, A. T.; Gordon, V. D.; Cristobal, G.; Talingting, M. R.; Bell, D. C.; Evans, C.; Joanicot, M.; Zasadzinski, J. A.; Weitz, D. A. Macromolecules 2004, 37, 2215. (16) Lim-Soo, P.; Eisenberg, A. J. Polym. Sci., Part B: Polym. Phys. 2004, 42, 923. (17) Solomatin, S. V.; Bronich, T. K.; Eisenberg, A.; Kabanov, V. A.; Kabanov, A. V. Langmuir 2004, 20, 2066. (18) Choucair, A.; Eisenberg, A. Eur. Phys. J. E 2003, 10, 37. (19) Yu, K.; Bartels, C.; Eisenberg, A. Langmuir 1999, 15, 7157.

aggregates offer the possibility of incorporation of guest molecules, which opens a wide range of potential applications, for example, in technical, medical, and pharmaceutical fields. In particular, in connection with the previously mentioned applications, extensive studies have been performed on the use of such structures, including micelles and vesicles, as carrier systems of various drugs. Micelles formed from amphiphilic di- or triblock copolymers have been explored as controlled drug delivery vehicles and targeting systems for hydrophobic drugs.21 Liposomes, or vesicles from lipid bilayers, play a significant role in pharmaceutical as well as cosmetic industries as biodegradable or biocompatible drug carriers, by enhancing the potency and reducing the toxicity of therapeutics.22 Block copolymer vesicles have been considered to be alternatives to liposomes.3,16,23 The advantage of the block copolymer vesicles over liposomes for some applications is their increased stability and the rigidity of their membranes,20 which contribute to their increased lifetime. The large number of available monomers and the ability to vary the ratio of the two constitutive blocks makes it possibile to tune the properties of the resulting nanostructures, for example, the aggregate dimensions, solubility, and stability. As compared to extensive research on the encapsulation of biomolecules into liposomes, the studies on encapsulation of large biological units into block copolymer vesicles are limited,24-26 in part, because large hydrophilic biomolecules are sensitive to organic solvents (20) Discher, B. M.; Won, Y.-Y.; Ege, D. S.; Lee, J. C.-M.; Bates, F. S.; Discher, D. E.; Hammer, D. A. Science (Washington, DC, U.S.) 1999, 284, 1143. (21) (a) Kataoka, K.; Kabanov, A. Colloids Surf., B 1999, 16 (entire volume). (b) Allen, C.; Maysinger, D.; Eisenberg, A. Colloids Surf., B 1999, 16, 3. (22) Lian, T.; Ho, R. J. Pharm. Sci. 2001, 90, 667. (23) Ding, J.; Liu, G. J. Phys. Chem. B 1998, 102, 6107. (24) Choucair, A.; Lim-Soo, P.; Eisenberg, A. Langmuir 2005, 21, 9308. (25) Korobko, A. V.; Jesse, W.; Maarel, J. R. C. Langmuir 2005, 21, 34.

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and the pH of the solution. The paucity of studies dealing with the encapsulation of biological species into block copolymer aggregates underlines the inherent difficulties of the process and suggests the need for further research. To improve the understanding of the encapsulation process of species comparable in size and function to biological materials as well as of the underlying mechanisms, we explored the supramolecular structure formation of a polystyrene-block-poly(acrylic acid) (PS-b-PAA) copolymer in the presence of a fourthgeneration amine-terminated, poly(amido amine) dendrimer (G4NH2 PAMAM),27 which are very useful model compounds for complex structures, such as proteins, which have similar sizes and/or similar terminal groups.28 The presence of carboxylic acid moieties on the block copolymer and of amine groups on the dendrimer, in a medium of relatively low polarity, such as dioxane, undoubtedly leads to strong interactions, which manifest themselves immediately upon mixing of the two components. The effect of such strong and specific interactions on the structure formation is of interest. It should be noted that the effect of polyelectrolyte complex formation on block copolymer selfassembly has been explored extensively.29 In those cases, however, the interactions occurred between flexible polyelectrolyte chains and are thus very different in their effect from those in the present system, which involves interactions between a relatively rigid sphere and a flexible polymer. The previous publication30 dealt with the effect of G4-NH2 PAMAM (at a constant amine/acid ratio of 0.06 or 1:16) on the hierarchical supramolecular structure formation of PS-b-PAA in a dioxane/THF/water solvent mixture.29 In the presence of G4NH2 PAMAM, the onset of self-assembly of single chains of PS-b-PAA (primary structure) into single and multiple dendrimer core inverse micelles MDCIMs (secondary structure) was detected by dynamic light scattering (DLS) at very low water contents of cW < 2 wt %. Because of the lower sensitivity of the turbidity method, the onset of self-assembly detected by turbidity measurements was found at a water content (cwc) of cW ) ∼7 wt %. The resulting micelles consisted of dendrimers coated by the PAA blocks, which are connected to the corresponding PS chains, which form the corona. Further addition of water led to an association of these hydrophobic micelles into compound multiple dendrimer core inverse micelles, CompMDCIMs (tertiary structure), in a range of cW ) ∼6 to ∼10 wt %. At still higher water contents, some of the acrylic acid chains of the block copolymer moved from the vicinity of the dendrimer to the outside (26) (a) Cornelissen, J. J. L. M.; Fischer, M.; Sommerdijk, N. A. J. M.; Nolte, R. J. M. Science (Washington, DC, U.S.) 1998, 280, 1427. (b) Brown, M. D.; Schatzlein, A.; Brownlie, A.; Jack, V.; Wang, L.; Tetley, L.; Gray, A. I.; Uchegbu, I. F. Bioconjugate Chem. 2000, 11, 880. (c) Lee, J. C.-M.; Bermudez, H.; Discher, B. M.; Sheehan, M. A.; Won, Y.-Y.; Bates, F. S.; Discher, D. E. Biotechnol. Bioeng. 2001, 73, 135. (d) Nardin, C.; Meier, W. ReV. Mol. Biotechnnol. 2002, 90, 17. (e) Napoli, A.; Boerakker, M. J.; Tirelli, N.; Nolte, R. J. M.; Sommerdijk, N. A. J. M.; Hubbell, J. A. Langmuir 2004, 20, 3487. (f) Brannan, A. K.; Bates, F. S. Macromolecules 2004, 37, 8816. (g) Mecke, A.; Dittrich, C.; Meier, W. Soft Matter 2006, 2, 751. (h) Wittemann, A.; Azzam, T.; Eisenberg, A. Langmuir 2007, 23, 2224. (27) Tomalia, D. A.; Naylor, A. M.; Goddard, W. A., III. Angew. Chem., Int. Ed. Engl. 1990, 29, 138. (28) (a) Uppuluri, S.; Keinath, S. E.; Tomalia, T. A.; Dvornic, P. R. Macromolecules 1998, 31, 4498. (b) Tomalia, D. A.; Huang, B.; Swanson, D. R.; Brothers, H. M., II; Klimash, J. W. Tetrahedron 2003, 59, 3799. (29) (a) Kataoka, K.; Togawa, H.; Harada, A.; Yasugi, K.; Matsumoto, T.; Katayose, S. Macromolecules 1996, 29, 8556. (b) Harada, A.; Kataoka, K. Macromolecules 1998, 31, 288. (c) Bronich, T. K.; Cherry, T.; Vinogradov, S. V.; Eisenberg, A.; Kabanov, V. A.; Kabanov, A. V. Langmuir 1998, 14, 6101. (d) Cohen Stuart, M. A.; Besseling, N. A.; Fokkink, R. G. Langmuir 1998, 14, 6846. (e) Kabanov, A. V.; Bronich, T. K.; Kabanov, V. A.; Yu, K.; Eisenberg, A. J. Am. Chem. Soc. 1998, 120, 9941. (f) Harada, A.; Kataoka, K. Science (Washington, DC, U.S.) 1999, 283, 65. (g) Berret, J.-F.; Cristobal, G.; Herve´, P.; Oberdisse, J.; Grillo, I. Eur. Phys. J. E 2002, 9, 301. (h) Geng, Y.; Ahmed, F.; Bhasin, N.; Discher, D. E. J. Phys. Chem. B 2005, 109, 3772. (30) Kroeger, A.; Li, X.; Eisenberg, A. Langmuir 2007, 23, 10732.

Li et al.

of the aggregates, resulting in a decrease of the size of the formed structures and the acquisition of progressively increasing hydrophilic character of the aggregates. Multiple dendrimer core inverse onion micelles, MDCIOMs, were formed, which agglomerated into compound multiple dendrimer core inverse onion micelles, CompMDCIOMs, at cW ) ∼12 to ∼18 wt %. Finally, above this water content, vesicles were formed. The level of complexity encountered here points out the importance of strong interactions as well as geometry in determining the morphology in block copolymer self-assembly and suggests that this field is worthy of further studies. The aim of the present work is, therefore, to elucidate the role of the ratio of amine groups of the dendrimers to acrylate units of the copolymer (RAm/Ac) on the self-organization of the system. The effects of different relative concentrations of dendrimer additives, as seen by turbidity measurements, are supplemented by direct imaging via transmission electron microscopy of the formed supramolecular aggregates, whose structure can be inferred by the combination of these methods. Thus, the distribution and localization of the dendrimer inside the formed aggregates, specifically vesicles, can be examined. As one aspect of this examination, some work was performed using dendrimers, which were modified with pyrenyl groups (G4-Py PAMAM), to allow fluorescence quenching studies. If the labeled dendrimers are contained within the formed block copolymer aggregates, and are thus protected by a continuous PS layer, as assumed, an external fluorescence quencher should not be able to quench the fluorescence.31 Furthermore, due to the modification, the size of the pyrenyl-labeled dendrimer G4-Py PAMAM increases as compared to that of the G4-NH2 PAMAM dendrimer, which offers the possibility of confirming the localization of the dendrimer within the aggregates by comparative size studies. Experimental Procedures Materials. The block copolymer aggregates were prepared from a polystyrene-block-poly (acrylic acid) copolymer, PS310-b-PAA36, where the numbers refer to the number-average degree of polymerization Pn. The details of the synthesis, characterization procedures, and sample preparation were given in previous publications.30,32 The solvents, 1,4-dioxane and tetrahydrofuran (THF, 99.9%), were purchased from Fisher Scientific; for all experiments, a fixed ratio of dioxane to THF of 75:25 wt % was used. A methanol solution of the fourth generation poly(amidoamine) dendrimer was obtained from Aldrich. The dendrimer consists of a tetra-functional core of ethylenediamine (>NCH2CH2N ∼15 wt %.

into the aggregates, as described previously. A G4-Py dendrimer containing a test solution of 0.5 wt % PS310-b-PAA36 in dioxane/ THF and a water content of cW ) 42.0 wt % was prepared by adding an aqueous G4-Py PAMAM solution of a concentration of cG4-PY PAMAM ) 1.7 × 10-6 g/mL. Therefore, a part of the pyrene groups is accessible for the quencher because this fraction is not integrated in the self-assembled structures, and, as indicated in Table 3, the fraction of accessible chromphores increases up to φ ) ∼0.25 ( 0.03, as assumed. Because, on all supramolecular hierarchical structure levels, specifically vesicles, the dendrimer is coated by the PAA chains of the block copolymer due to strong interactions (as proven by the fluorescence quenching studies), and the PAA blocks are connected to the PS blocks, which form the corona, PAMAM is surrounded by PS chains and thus encapsulated into the micelles or in the vesicle walls. Consequently, the precise vesicle structure may be described (going outward) as a multilayer system (i.e., PAA (inner corona)-PS-encapsulated dendrimer units-PSPAA (outer corona)), as illustrated in Figure 7 for RAm/Ac ) 0.06. It should be stressed again that, as indicated by Figure 2, with

Structure Formation of Block Copolymers

increasing RAm/Ac values, more and more individual dendrimer cores are encapsulated in the formed constitutive structures (as described previously) and, consequently, in the vesicle walls, and therefore, the wall thicknesses increase with increasing dendrimer concentration. Furthermore, this gives the possibility of a targeted loading of species comparable in size and function to biological materials into the vesicle walls and an additional loading of the interior of these structures.

Conclusion In this work, the dendrimer concentration dependence of the supramolecular structure formation of polystyrene-block-poly(acrylic acid) (PS-b-PAA) was investigated. The solvent was a mixture of dioxane/THF (fixed ratio of 75:25 wt %), and the studies were performed as a function of the water content by using turbidity measurements and TEM. The supramolecular structure formation of PS-b-PAA in the absence as well as in the presence of G4-NH2 PAMAM, for RAm/Ac ) 0.06, was reported in the previous publication.30 In the present study, the turbidity profiles of all dendrimer-containing solutions (RAm/Ac > 0) show considerable similarity, which suggest a similarity in the supramolecular structures. Over the range of RAm/Ac ) 0.030.24, the structure formation is qualitatively similar; quantitatively, the sizes of the formed aggregates increase with increasing RAm/Ac. In view of the strong interactions between the participating species, the structural similarity over this large concentration range could not be predicted a priori. One factor, which changes with the RAm/Ac value, is the number of dendrimer units in the MDCIMs. It appears that the multiple dendrimer core inverse micelles

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(MDCIMs) are very robust structures because of the present strong interactions and probably survive intact the changes in morphology as function of the water content. As was seen in the previous publication,30 the morphological progression as a function of water content goes from dendrimer core inverse micelles to vesicles. In the last mentioned case, the sizes of the vesicles as well as their wall thickness are influenced by RAm/Ac: both increase with increasing dendrimer concentration. Thus, at low RAm/Ac values, the increase in vesicle wall thickness is moderate but increases with increasing RAm/Ac values in parallel with the increase in the number of dendrimer cores in the inverse micelles. The formed dendrimer core inverse micelles are incorporated in the wall of vesicles. The vesicle wall may be described as a multilayer system. The proposed structure of the formed aggregates as a function of cW is confirmed by fluorescence quenching studies and in one case also by a high-resolution TEM image, which shows the localization of the dendrimer cores in the center of the micelles. Acknowledgment. For the high-resolution TEM micrograph, we are grateful to Dr. I. Lieberwirth (Max Planck Institute for Polymer Research, Mainz, Germany). We thank the National Science and Engineering Research Council of Canada for continuing support of this research. Supporting Information Available: Details of synthesis and characterization of G4-Py PAMAM as well as fluorescence spectra of aqueous G4-Py PAMAM solutions. This material is available free of charge via the Internet at http://pubs.acs.org. LA702614X