pubs.acs.org/Langmuir © 2009 American Chemical Society
Aggregation Behavior of Graft Copolymer with Rigid Backbone Chunhua Cai, Jiaping Lin,* Tao Chen, and Xiaohui Tian Shanghai Key Laboratory of Advanced Polymeric Materials, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237, China Received August 1, 2009. Revised Manuscript Received September 22, 2009 The self-assembly behavior of poly(γ-benzyl-L-glutamate)-graft-poly(ethylene glycol) rod-coil graft copolymers in aqueous solution was investigated. With tetrahydrofuran (THF) as initial solvent, vesicles were observed for the graft copolymers with lower degree of grafting. When the degree of grafting increases, the aggregate morphology transforms from vesicles to spindle-like micelles then to spherical micelles. When N,N0 -dimethylformamide (DMF) is introduced into the initial solvent, the vesicles transform to spindles. Increasing DMF volume fraction leads to a spindle to connected-spindle transition. On the basis of the experimental results, the mechanism of the morphological transition of the rod-coil graft copolymer is suggested.
Introduction Polymeric micelles, self-assembled from amphiphilic block copolymers in selective solvents, are of intensive interest in contemporary macromolecular science for both their diversiform morphologies and potential applications.1-9 Graft copolymers are another important building block forming self-assembled aggregates, though they have obtained much less attention as compared with the block copolymers. However, they have obvious advantages in adjusting the self-assembly behavior by changing the side-chain properties, such as grafting density, chain length, environmental sensitivity, etc.10-23 Most of the block or graft copolymers studied so far are coil-coil type copolymers. Introducing rigid segment into the block copolymers makes them exhibit much more distinct self-assembly features *Corresponding author: Tel þ86-21-64253370; Fax þ86-21-64253539; e-mail
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than those of the coil-coil type block copolymers because the rigid blocks prefer to take ordered packing mode during aggregation.24-29 However, there are only a few works on the self-assembly behavior of the graft copolymers (or graft-like polymers) bearing a rigid segment.30-34 Xia et al. have studied the self-assembly behavior of perfluorosulfonated ionomer (Nafion) in dilute aqueous solutions and in water-based mixed solvents.30 It was found that the fluorocarbon backbone forms the core of the rod-like micelle, and the pendant side chain with an ionic group shields the core of the micelle. Jo’s group has studied the micellization behavior of rod-coil graft copolymers in solution using Brownian dynamics simulations.35 It was revealed that the hydrophobic rigid backbones tend to pack with each other to form cylindrical micelles stabilized by outspreaded hydrophilic side segments. Recently, increasing attention has been given to the polypeptidebased self-assemblies due to their excellent biocompatibility and significant advantages in controlling both the function and structures of the supramolecular aggregates.36-42 Lecommandoux (24) Jenekhe, S. A.; Chen, X. L. Science 1998, 279, 1903. (25) Jenekhe, S. A.; Chen, X. L. Science 1999, 283, 372. (26) Klok, H.-A.; Lecommandoux, S. Adv. Mater. 2001, 13, 1217. (27) Lee, M.; Cho, B.-K.; Zin, W.-C. Chem. Rev. 2001, 101, 3869. (28) Pinol, R.; Jia, L.; Gubellini, F.; Levy, D.; Albouy, P.-A.; Keller, P.; Cao, A.; Li, M.-H. Macromolecules 2007, 40, 5625. (29) Yang, J.; Levy, D.; Deng, W.; Keller, P.; Li, M.-H. Chem. Commun. 2005, 4345. (30) Jiang, S.; Xia, K.-Q.; Xu, G. Macromolecules 2001, 34, 7783. (31) Yao, J. H.; Mya, K. Y.; Shen, L.; He, B. P.; Li, L.; Li, Z. H.; Chen, Z.-K.; Li, X.; Loh, K. P. Macromolecules 2008, 41, 1438. (32) Wilhelm, V.; Hellman, G. P. Polymer 2000, 41, 1905. (33) Wang, C.; Li, G.; Guo, R. Chem. Commun. 2005, 3591. (34) Liao, S.-C.; Lai, C.-S.; Yeh, D.-D.; Rahman, M. H.; Hsu, C.-S.; Chen, H.-L.; Chen, S.-A. React. Funct. Polym. 2009, 69, 498. (35) Kim, K. H.; Huh, J.; Jo, W. H. Macromolecules 2004, 37, 676. (36) Carlsen, A.; Lecommandoux, S. Curr. Opin. Colloid Interface Sci. 2009, 14, 329. (37) Rodrı´ guez-Hernandez, J.; Lecommandoux, S. J. Am. Chem. Soc. 2005, 127, 2026. (38) Checot, F.; Lecommandoux, S.; Gnanou, Y.; Klok, H.-A. Angew. Chem., Int. Ed. 2002, 41, 1339. (39) Rao, J.; Luo, Z.; Ge, Z.; Liu, H.; Liu, S. Biomacromolecules 2007, 8, 3871. (40) Sun, J.; Chen, X.; Deng, C.; Yu, H.; Xie, Z.; Jing, X. Langmuir 2007, 23, 8308. (41) Wong, M. S.; Cha, J. N.; Choi, K.-S.; Deming, T. J.; Stucky, G. D. Nano Lett. 2002, 2, 583. (42) Iatrou, H.; Frielinghaus, H.; Hanski, S.; Ferderigos, N.; Ruokolainen, J.; Ikkala, O.; Richter, D.; Mays, J.; Hadjichristidis, N. Biomacromolecules 2007, 8, 2173.
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et al. have explored self-assembly behavior of poly(L-glutamic acid) (PLGA)- and poly(L-lysine) (PLL)-based amphiphilic diblock copolymers in aqueous solution.37,38 It was found that vesicles or spherical micelles are formed with different block ratio, and a change of solution pH value has marked impact on the aggregate size. However, there is few study concerning the selfassembly behaviors of polypeptide-based graft copolymers.43-48 Recently, our group has studied the self-assembly behavior of polypeptide-based block and graft copolymers.49-53 The graft copolymer consists of a rigid poly(γ-benzyl-L-glutamate) (PBLG) backbone, on which hydrophilic poly(ethylene glycol) (PEG) side chains are grafted.52,53 The rod-coil graft copolymers were found to associate into spindle-like micelles in solutions. The rigid polypeptide blocks align with each other inside the micelle core, while PEG chains surround outside to stabilize the aggregates. However, the understanding of the influence of molecular structure on the self-assembly of the PBLG-g-PEG samples is still not clear enough. The initial common solvent is an important factor influencing the self-assembly behavior of block copolymers. For example, Eisenberg’s group has prepared various morphologies from highly asymmetric diblock copolymers; the aggregate morphology can be tuned by the nature of initial solvent.1-3 However, for graft copolymers, the study of the initial solvent nature on their self-assembly behavior is rare. It is necessary to explore the influence of initial solvent nature on the micellization of rod-coil type graft copolymers. Herein, we report the self-assembly behavior of rod-coil PBLG-g-PEG graft copolymers as functions of the degree of grafting and initial commom solvent nature. It was found, for the first time, that graft copolymers with rigid polypeptide backbone form vesicles. The vesicles, which are formed through dialysis against water with tetrahydrofuran (THF) as initial solvent, were found to transform to spindles then to spheres when the degree of grafting increases. In addition, the vesicles transformed to spindles when N,N0 -dimethylformamide (DMF) was added to the initial solvent. Increasing DMF volume fraction leads to a transition of aggregate morphology from spindle to connectedspindle. On the basis of the experimental results, the mechanism of the morphological transition of the rod-coil graft copolymer aggregates is suggested.
Experimental Section Materials. Poly(ethylene glycol) monomethyl ether (mPEG) (Mw = 750) was purchased from Sigma Inc. and used without further purification. The dialysis bag (Membra-cel, 3500 molecular weight cutoff) was provided by Serva Electrophoresis GmbH. Analytical grades of hexane, tetrahydrofuran (THF), and 1,4-dioxane were refluxed with sodium and distilled immediately before use. N,N0 -Dimethylformamide (DMF) was distilled (43) Jun, Y. J.; Toti, U. S.; Kim, H. Y.; Yu, J. Y.; Jeong, B.; Jun, M. J.; Sohn, Y. S. Angew. Chem., Int. Ed. 2006, 45, 6173. (44) Konak, C.; Reschel, T.; Oupicky, D.; Ulbrich, K. Langmuir 2002, 18, 8217. (45) Nottelet, B.; El Ghzaoui, A.; Coudane, J.; Vert, M. Biomacromolecules 2007, 8, 2594. (46) Xu, N.; Du, F.-S.; Li, Z.-C. J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 1889. (47) Jeong, J. H.; Kang, H. S.; Yang, S. R.; Kima, J.-D. Polymer 2003, 44, 583. (48) Chen, W.; Chen, H.; Hu, J.; Yang, W.; Wang, C. Colloids Surf., A 2006, 278, 60. (49) Cai, C.; Lin, J.; Chen, T.; Wang, X.-S.; Lin, S. Chem. Commun. 2009, 2709. (50) Cai, C.; Zhang, L.; Lin, J.; Wang, L. J. Phys. Chem. B 2008, 112, 12666. (51) Ding, W.; Lin, S.; Lin, J.; Zhang, L. J. Phys. Chem. B 2008, 112, 776. (52) Tang, D.; Lin, J.; Lin, S.; Zhang, S.; Chen, T.; Tian, X. Macromol. Rapid Commun. 2004, 25, 1241. (53) Lin, J.; Zhu, G.; Zhu, X.; Lin, S.; Nose, T.; Ding, W. Polymer 2008, 49, 1132.
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Scheme 1. Synthesis and Structure of PBLG-g-PEG Graft Copolymer
under reduced pressure and stored over activated 4 A˚ molecular sieves. All the other regents are of analytical grade and used as received.
Synthesis of PBLG Homopolymer and PBLG-g-PEG Graft Copolymers. γ-Benzyl-L-glutamate-N-carboxyanhydride (BLG-NCA) was synthesized according to the literature procedures.54 PBLG was obtained by the ring-opening polymerization of BLG-NCA initiated by triethylamine with 1,4-dioxane as solvent.54,55 After reacted at room temperature for 72 h, the viscous reaction mixture was poured into a large volume of anhydrous ethanol. The precipitated product was dried under vacuum and then purified twice by repeated precipitation from a chloroform solution into a large volume of anhydrous methanol. The molecular weight of the PBLG sample was estimated to be 171 000 from the intrinsic viscosity ([η]) value measured in dichloroacetic acid (DCA). The gel permeation chromatography (GPC) analysis (Waters 1515, DMF as eluent solvent) shows that the molecular weight distribution of the PBLG homopolymer is 1.18. As shown in Scheme 1, PBLG-g-PEG graft copolymers were prepared by the ester exchange reaction of PBLG with mPEG.52,53,56,57 The reaction was performed at 55 C in 1,2dichloroethane (DCE) with p-toluenesulfuric acid (TSA) as a catalyst. Then the reaction mixture was precipitated into a large volume of anhydrous methanol. The product was purified twice by repeated precipitation from a chloroform solution into a large volume of anhydrous methanol and then dried under vacuum. The degree of grafting of PBLG-g-PEG graft copolymer was calculated by the peak intensities of the methylene proton signal (5.1 ppm) of PBLG and the ethylene proton signal (3.6 ppm) of PEG in the 1H NMR spectrum.49,50 By the variation of molar ratio of BLG unit to mPEG and the reaction time, the degree of grafting could be adjusted. Finally, three graft copolymers denoted as PBLG-gPEG1, PBLG-g-PEG2, and PBLG-g-PEG3 were obtained. Calculations showed that the degree of grafting is 0.28, 1.5, and 5.2 mol %, respectively. Detailed information regarding the synthesis and characteristics of the graft copolymers is provided in Table 1. Preparation of Micelles. The polymeric micelle solutions were prepared using a dialysis method. The obtained PBLG-gPEG graft copolymers were first dissolved in THF, DMF, or their mixed solvent. The initial polymer concentration (C) was 0.25 g/L. Then 2.5 mL of deionized water, a selective solvent for PEG, was added to 10 mL of polymer solution at a rate (R) of 0.02 mL/s with vigorous stirring. Next the solution was dialyzed against deionized water for 3 days at room temperature. Before analysis, the solutions were stabilized for at least 5 days. 1 H NMR. The degree of grafting of the graft copolymers was obtained by NMR measurements. 1H NMR spectrum measurements were performed on a Bruker Advance 500 spectrometer (54) Blout, E.; Karlson, R. J. Am. Chem. Soc. 1956, 78, 941. (55) Lin, J.; Abe, A.; Furuya, H.; Okamoto, S. Macromolecules 1996, 29, 2584. (56) Inomata, K.; Ohara, N.; Shimizu, H.; Nose, T. Polymer 1998, 39, 3379. (57) Inomata, K.; Shimizu, H.; Nose, T. J. Polym. Sci., Part B: Polym. Phys. 2000, 38, 1331.
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Article Table 1. Details of Synthesis and Characteristics of PBLG-g-PEG Graft Copolymers
sample
feed molar ratio of mPEG to BLG
reaction time (h)
degree of grafting (mol %)
EG content (mol %)
PEG weight fraction (vol %)
PBLG-g-PEG1 PBLG-g-PEG2 PBLG-g-PEG3
0.05 0.2 0.2
1 2 5
0.28 1.5 5.2
4.40 20.1 47.2
0.95 4.81 15.1
using deuterated chloroform (CDCl3) as solvent and tetramethylsilane (TMS) as an internal standard at room temperature. The concentration of the polymers in all the solutions is ca. 0.5 wt %. Circular Dichroism (CD). CD analyses of the conformation of polypeptide segments were performed with a JASCO J810 spectrometer at room temperature with THF as solvent. The diluted solutions (0.10 g/L for all the samples) were introduced in quartz cells with 1 cm optical path length. Wavelengths between 200 and 300 nm were analyzed, with an integration time of 1 s and a wavelength step of 0.2 nm. The molar ellipticity [θ] of the polymers can be calculated according to the following equation: [θ] = θ/(10ML), where θ, M, and L are the absorption value of polymer solutions (mdeg), molar concentration of the peptide units (mol L-1), and optical path length of quartz cell (cm), respectively. In addition, from the value of [θ] the helix content of the polymers can be estimated (helix content [%] = ([θ]/[θ]h) 100%, where [θ]h is -34 000 deg cm2 dmol-1).58 Transmission Electron Microscopy (TEM). The morphologies of the aggregates were examined by TEM (JEM-2000EXII, JEOL) operated at an accelerating voltage of 60 kV. Drops of solution were placed on a copper grid coated with carbon film and then were dried at room temperature. Before the observations, the samples were stained by phosphotungstic acid aqueous solution (0.5 wt %). Scanning Electron Microscopy (SEM). The morphologies of the aggregates were also observed by SEM (JSM 6460, JEOL) operated at an accelerating voltage of 20 kV. The samples were prepared by placing drops of solution on a copper grid coated with carbon film and then were dried at room temperature. Before the observations, the samples were sputtered by carbon. Light Scattering Measurements (LLS). Laser light scattering was measured by a LLS spectrometer (ALV/CGS-5022) equipped with an ALV-High QE APD detector and an ALV5000 digital correlator using a He-Ne laser (the wavelength λ = 632.8 nm) as the light source. All the measurements were carried out at 20 C. In static LLS, the angular dependence of the excess absolute time-average scattered intensity, i.e., Rayleigh ratio Rvv(q) of the dilute polymer solutions was measured. Rvv(q) is related to the weight-average molar mass (Mw), polymer concentration (C), and the scattering angle (j) as KC=Rðj, CÞ ¼ 1=Mw ½1 þ ðRg 2 q2 Þ=3 þ 2A2 C
ð1Þ
where K = 4π n (dn/dC) /(NAλ ) and q = 4πn sin(j/2)/λ with NA, dn/dC, n, and λ being the Avogadro number, the specific refractive index increment, the solvent refractive index, and the wavelength of the light in vacuum, respectively, A2 is the second virial coefficient, and Rg is the z-average radius of gyration of the aggregates in solution. By extrapolating to zero concentration and zero angle, Rg values of the aggregates can be calculated. In dynamic LLS measurement, the Laplace inversion of each measured intensity-intensity time correlation function G(2)(t,q) in the self-beating mode can result in a line-width distribution G(Γ). The translational diffusion coefficient D calculated from the decay time, Γ, by the slope of the Γ vs q2 plot can lead to 2 2
2
4
(58) Higashi, N.; Koga, T.; Niwa, M. Adv. Mater. 2000, 12, 1373.
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hydrodynamic radius Rh by the Stokes-Einstein equation Rh = kBT/(6πηD), where kB, T, and η are the Boltzmann constant, the absolute temperature, and the solvent viscosity, respectively.
Results and Discussion The PBLG-g-PEG copolymer consists of a rigid polypeptide backbone, on which hydrophilic PEG side chains are grafted (see Scheme 1). The graft copolymer dissolves well in THF, DMF, or their mixed solvent. Upon the addition of water, a strong selective solvent for PEG segment, the solubility of mixed solvent (THF/DMF/H2O) for PEG segments remains well but becomes worse for polypeptide segments. With gradually increasing water content, the hydrophobic polypeptide segments becomes insoluble and tend to form aggregate core outspreaded with PEG chains. Finally, through dialysis process, the structures were frozen, and water-dispersed aggregate solutions were obtained. Because of the good solubility of PEG segments in water,59,60 the formed aggregates are stable in aqueous solution (see Figure S1 in the Supporting Information). In this work, the influences of the degree of grafting and initial solvent composition on the self-assembly behavior of PBLG-g-PEG graft copolymers were investigated. Morphologies Observed by Microscopies. Figure 1 shows the typical TEM photographs of aggregate morphologies formed by graft copolymers as functions of degree of grafting and initial solvent composition. The degree of grafting has a significant effect on the aggregate morphology. Taking the samples prepared with pure THF as initial common solvent for example, when the degree of grafting is lower, vesicles are formed (PBLG-g-PEG1, Figure 1a). The vesicles have a uniform diameter and thickness. The ring-like images observed by TEM is a typical feature of hard vesicles.61 Because of the rigid structure of wall formed by PBLG blocks, the vesicles may not collapse completely under high vacuum in TEM observations. The TEM photo is a projection image of vesicles. Thus, the electron contrast is very strong from edge to center in TEM observations; as a result, ring-like images were observed from TEM. As the degree of grafting increases, the aggregate morphology transforms to spindle-like micelles (PBLG-g-PEG2, Figure 1d) and then to spherical micelles (PBLG-g-PEG3, Figure 1g). The initial common solvent nature also affects the aggregation behavior of the graft copolymers. For PBLG-g-PEG1, the aggregate morphology transforms from vesicles (Figure 1a) to spindles (Figure 1b) and then to connected-spindles (Figure 1c), when the volume fraction of DMF (fDMF) in initial solvent increases from 0 to 0.5 and then to 1.0. It is noted that concomitantly with the transition from spindle to connectedspindle the diameter of the aggregates become smaller. This obviously suggests that the interfacial area between the PBLG (59) Otsuka, H.; Nagasaki, Y.; Kataoka, K. Curr. Opin. Colloid Interface Sci. 2001, 6, 3. (60) Dunn, S. E.; Brindley, A.; Davis, S. S.; Davies, M. C.; Illum, L. Pharm. Res. 1994, 11, 1016. (61) Yang, M.; Wang, W.; Yuan, F.; Zhang, X.; Li, J.; Liang, F.; He, B.; Minch, B.; Wegner, G. J. Am. Chem. Soc. 2005, 127, 15107.
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Figure 2. SEM photographs of vesicles self-assembled from PBLG-g-PEG1 graft copolymers with pure THF as initial solvent. The inset is a high-magnification image.
Figure 1. TEM images of aggregates self-assembled from PBLGg-PEG graft copolymers as functions of degrees of grafting and THF/DMF compositions in initial common solvents: PBLG-gPEG1, with fDMF = 0 (a), 0.50 (b), and 1.0 (c); PBLG-g-PEG2, with fDMF = 0 (d), 0.50 (e), and 1.0 (f); PBLG-g-PEG3, with fDMF = 0 (g), 0.50 (h), and 1.0 (i). The initial copolymer concentration (C) is 0.25 g/L, and the adding rate of water (R) is 0.02 mL/s.
core and PEG shell increases. For the PBLG-g-PEG2 sample, as fDMF increases in the initial solvent, the aggregate morphology transforms from spindles (fDMF = 0, Figure 1d) to connectedspindles (fDMF = 0.5, Figure 1e). Further increase in fDMF leads to connected-spindles with a smaller diameter (fDMF = 1.0, Figure 1f). The highly grafted PBLG-g-PEG3 self-assembles into spheres in all the composition of initial solvent. However, the size of the spheres decreases slightly, when fDMF is increased (Figure 1g-i). Because of the long chain nature, polymers have very low mobility in solution; therefore, in the micellization process they are easy to form kinetically trapped structures from different initial conditions (these structures are frozen and exist in metastable state). As a result, different preparation methods such as different common solvents could give rise to different metastable structures. However, these structures are stable for times. Such phenomena are general to polymer systems and widely reported in the literature.3,62,63 The observed vesicular structure was further examined using SEM and AFM analyses. Shown in Figure 2 is a typical SEM image of the vesicles, which gives a three-dimensional shape. The magnified image in Figure 2 clearly shows a lower center on the surface of the vesicle. Because of the rigid structure of wall formed by PBLG segments, under the very high vacuum during SEM analysis, the vesicles do not collapse completely, but collapse on the top surface of the aggregates. AFM testing also proves the vesicular structure of the aggregate, as shown in Figure 3. The high periphery and low center of the aggregates indicate the periphery and the center of vesicles have different responses to the force of the AFM probe.40,61 Note that the diameter of the vesicles is slightly larger in AFM image than that in TEM and SEM (62) Cui, H.; Chen, Z.; Zhong, S.; Wooley, K. L.; Pochan, D. J. Science 2007, 317, 647. (63) Yu, Y.; Zhang, L.; Eisenberg, A. Macromolecules 1998, 31, 1144.
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Figure 3. AFM image of vesicles self-assembled from PBLG-gPEG1 graft copolymers with pure THF as initial solvent.
Figure 4. TEM images of aggregates self-assembled from PBLGg-PEG1 graft copolymers with different methods: (a) accelerating the adding rate of water (R = 0.2 mL/s) and (b) diluting the initial copolymer concentration (C = 0.05 g/L).
images. The vesicles are collapsed under high vacuum of TEM and SEM observations. However, the AFM testing was carried out under atmospheric pressure, and the vesicles shrink slightly. In the experiments, we also find that the adding rate of selective solvent and the initial copolymer concentration influence the micellization of highly asymmetric PBLG-g-PEG graft copolymers. Figure 4 gives the TEM graphs of aggregates self-assembled from PBLG-g-PEG1 with (a) accelerating the adding rate of selective solvent (change from 0.02 to 0.2 mL/s) and (b) diluting the initial copolymer concentration (change from 0.25 to 0.05 g/L). It was found that accelerating the adding rate of selective solvent minishes the size and thickness of the vesicles and broadens the particle size distribution, as shown in Figure 4a. When the initial polymer solution was diluted, the aggregate morphology transformed from vesicles to a mixture of vesicles and spindles Langmuir 2010, 26(4), 2791–2797
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Figure 7. Typical Zimm plot of vesicles prepared from PBLG-gPEG1 graft copolymers with pure THF as initial solvent. Figure 5. PBLG-g-PEG graft copolymers aggregate morphology stability regions as functions of grafting ratio and composition of initial solvent. In addition to PBLG-g-PEG1, -2, and -3, data of PBLG-g-PEG graft copolymers with degrees of grafting of 0.55 and 3.2 mol % are included in the morphology diagram.
Table 2. Typical LLS Results of Self-Assembled Aggregates sample
fDMFa
ÆRhæ/nm
ÆRgæ/nm
ÆRgæ/ÆRhæ
PDIb
PBLG-g-PEG1 0 155.2 162.3 1.03 0.07 PBLG-g-PEG1 0.5 180.1 291.3 1.62 0.11 PBLG-g-PEG1 1.0 254.6 374.3 1.47 0.13 PBLG-g-PEG2 0 108.9 163.4 1.50 0.08 PBLG-g-PEG2 1.0 215.4 306.7 1.42 0.14 PBLG-g-PEG3 0 77.1 58.2 0.75 0.07 PBLG-g-PEG3 1.0 71.3 52.3 0.73 0.08 a The volume fraction of DMF in initial THF/DMF mixed solvent. b Polydispersity index (PDI) of the aggregates was determined at the scattering angle of 60.
Figure 6. Plots of ÆRhæ vs volume fraction of DMF (fDMF) in initial solvent for PBLG-g-PEG graft copolymers with various degrees of grafting.
(Figure 4b). Both the vesicles and spindles are smaller in size. Increasing the adding rate of selective solvent makes polymer chains being quickly frozen in the primary formed aggregates; the chain exchange between aggregates is prevented (the formed aggregates are kinetically trapped to metastable state).64 When the initial copolymer concentration is diluted, less polymer chains can get into primary formed aggregates before they are frozen by adding enough selective solvent. As a result, both accelerating the adding rate of selective solvent and diluting the initial copolymer concentration decrease the aggregation number of micelles, which results in a decrease of aggregate size and/or a variation in the aggregate morphology.2,3,65,66 On the basis of the microscopic observations, we plot the aggregate morphology stability regions as functions of degree of grafting and the initial solvent composition, which is shown in Figure 5. The phase diagram is divided into four characteristic zones: vesicles, spindles, connected-spindles, and spheres. Vesicles were observed for the copolymers with lower degree of grafting and lower fDMF in initial solvent. Spheres were formed from the copolymers with higher degree of grafting or with moderate degree of grafting at lower fDMF in initial solvent. In other cases, spindles or connected-spindles were produced. Aggregate Size and Structure Studied by LLS. The aggregate structure of PBLG-g-PEG graft copolymers were further characterized by LLS. Figure 6 shows the hydrodynamic radius (ÆRhæ) variation of the aggregates as a function of initial common (64) Cao, H.; Lin, W.; Liu, A.; Zhang, J.; Wan, X.; Zhou, Q. Macromol. Rapid Commun. 2007, 28, 1883. (65) Alexandridis, P.; Holzwarth, J. F.; Hatton, T. A. Macromolecules 1994, 27, 2414. (66) Zhang, L.; Eisenberg, A. Macromolecules 1999, 32, 2239.
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solvent composition. Increasing fDMF in initial solvent makes the ÆRhæ value of PBLG-g-PEG1 increase markedly from about 160 nm to about 235 nm, which corresponds to the aggregate morphology transition from vesicles to spindles and connectedspindles. A similar phenomenon was obtained for PBLG-gPEG2; that is, the ÆRhæ value increases from about 110 nm to about 215 nm. However, the composition of the initial solvent has weak influence on the self-assembly behavior of PBLG-g-PEG3; the ÆRhæ decreases slightly as fDMF increases. These light scattering results are in good agreement with TEM observations. In addition, LLS testing shows that the formed aggregates are stable in solution for a long time (see Figure S1 in the Supporting Information). The change of aggregate structure can be also viewed in terms of the ratio of the radius of gyration (ÆRgæ to ÆRhæ), which is sensitive to the particle shape.67-69 Generally, ÆRgæ/ÆRhæ=0.774 is regarded as a uniform and nondraining sphere. When ÆRgæ/ ÆRhæ = 1, it can be attributed to a vesicle geometry in theory. For nonspherical structures, the ÆRgæ/ÆRhæ usually has a large value. Figure 7 is a typical Zimm plot of vesicles self-assembled from PBLG-g-PEG1 with THF as initial solvent. It gives a ÆRgæ value of 162.3 nm. Combining the ÆRhæ value (155.2) of the aggregates, the ÆRgæ/ÆRhæ value (1.04) is obtained. Typical LLS results of the selfassembled aggregates are summarized in Table 2. One can see that the ÆRgæ/ÆRhæ values of the vesicles (PBLG-g-PEG1, fDMF = 0) and spheres (PBLG-g-PEG3, fDMF = 0 and 1.0) are about 1 and 0.75, respectively, which are very close to the ideal ones. As for the spindles and connected-spindles, the ÆRgæ/ÆRhæ value is relatively large (for example, PBLG-g-PEG1, fDMF = 1.0, ÆRgæ/ÆRhæ = 1.47). These are reasonable results for rod-like structures, since the rods usually have larger ÆRgæ/ÆRhæ values.67-69 Additionally, the LLS results show that the self-assembled aggregates are narrowly distributed as indicated by the small polydispersity index (PDI). Typical PDI data are presented in (67) Chu, B. In Laser Light Scattering, 2nd ed.; Academic Press: New York, 1991. (68) Wu, C.; Li, M.; Kwan, S.; Liu, G. Macromolecules 1998, 31, 7553. (69) Vagberg, L. J. M.; Cogan, K. A.; Gast, A. P. Macromolecules 1991, 24, 1670.
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Figure 8. ÆRhæ distribution of the aggregates self-assembled from PBLG-g-PEG1 as the functions of initial copolymer concentration (C) and adding rate of water (R): (a) C = 0.25 g/L, R = 0.02 mL/s; (b) C = 0.25 g/L, R = 0.2 mL/s; and (c) C = 0.05 g/L, R = 0.02 mL/s. The scattering angle is 90.
Figure 9. CD spectra of homo-PBLG and PBLG-g-PEG graft copolymers in THF.
Table 2. For PBLG-g-PEG1, with the aggregate morphology transforms from vesicle to spindle than to connected-spindle, the PDI value increases slightly from 0.07 to 0.13. Similarly, the PDI value of PBLG-g-PEG2 aggregates changes slightly when the aggregate morphology transforms from spindle to connectedspindle. As PBLG-g-PEG3 forms uniform spherical micelles in all the composition of initial solvent, the PDI value keeps small (around 0.07). The preparation method also affects the PDI value of the aggregates. Shown in Figure 8 is the ÆRhæ distribution of the aggregates self-assembled from PBLG-g-PEG1 with different initial copolymer concentration (C) and adding rate of water (R). It was found that, with accelerating the adding rate of water or diluting the initial copolymer concentration, the ÆRhæ of the aggregates decreases and the PDI value increases. These results are in good agreement with TEM observations shown in Figure 4. Conformation Analysis of Homo-PBLG and PBLG-gPEG Graft Copolymers in Solution. It is well-known that PBLG adopts R-helix conformation in helicogenic solvents, such as THF, DMF, etc. However, the influence of grafting on the conformation of PBLG backbone is yet unclear. In this work, the influence of grafting on the secondary structure of PBLG backbone is characterized. Figure 9 shows the CD spectra of homoPBLG and PBLG-g-PEG graft copolymers in THF (DMF cannot be applied as the solvent for the CD analysis due to the strong absorption of amide bond). The spectra show a negative minimum at 228 nm, indicating that the polypeptide adopts the R-helix structure.70,71 It is noted that the wavelength of this peak is longer than the typical value of 222 nm reported for PBLG.72 This band is widely reported to be assigned to the n-π* transition of (70) Losik, M.; Kubowicz, S.; Smarsly, B.; Schlaad, H. Eur. Phys. J. E 2004, 15, 407. (71) Chung, T. W.; Cho, K. Y.; Nah, J. W.; Akaike, T.; Cho, C. S. Langmuir 2002, 18, 6462. (72) Cho, C.-S.; Nah, J.-W.; Jeong, Y.-I.; Cheon, J.-B.; Asayama, S.; Ise, H.; Akaike, T. Polymer 1999, 40, 6769.
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Figure 10. 1H NMR spectra of PBLG-g-PEG2 graft copolymers in THF-d8 and DMF-d7.
peptide bonds, which can be influenced by many factors such as solvent, side chain, and intra- or intermolecular interactions.73,74 Actually, a perfect CD spectrum consists of two negative minima at 208 and 222 nm were detected for PBLG-g-PEG micelle aqueous solution, which proves the R-helix conformation of PBLG (see Figure S2 in the Supporting Information). From the CD spectra one can estimate that all the polymers have an almost same helix content of ca. 90% in THF;70 the grafted PEG segments have a very weak influence on the secondary structure of the polypeptide. These CD results agree with the inherent character of the polypeptides in helicogenic solvents; that is, the polypeptide takes an imperfect helix in solution and contains a few units that are not incorporated in R-helix sequences.75 This makes the R-helix depart from rigid rectilinearity. The polypeptides act as broken rods and show flexibility to some extent. It is reported that the R-helix content of polypeptides differs slightly in different helicogenic solvents.76 In order to examine the influence of the solvent on the conformation of homo-PBLG and PBLG-g-PEG copolymers, 1H NMR analysis was applied. Figure 10 shows the typical 1H NMR spectra of homo-PBLG and PBLG-g-PEG2 graft copolymers in THF-d8 and DMF-d7. The peaks in 4.1 and 4.6 ppm are typical signals for R-helix and random coil conformation of R-H in polypeptide backbones, respectively. As revealed in Figure 10, in both THF and DMF, the polypeptides mainly take R-helix conformation, and the grafting has a less marked effect on the conformation of PBLG backbone. But the coil conformation content of the polypeptides in DMF is slightly higher than in THF, when carefully comparing Figure 10a,c with Figure 10b,d. (An enlarged version of the NMR spectra is provided in Figure S3 in the Supporting Information, which gives a more clear indication.) These results indicated that interaction between PBLG and DMF is relatively stronger than that between PBLG and THF. In DMF, more intramolecular H-bonding of PBLG backbone is destroyed by solvent, and the helix content of polypeptide is slightly lower than in THF. This is because both PBLG and DMF have amide bond, which gives rise to a relatively stronger interaction.77,78 In addition to the stronger interaction between PBLG backbone and DMF, the PBLG side benzyl groups also have stronger interaction with DMF than THF. The side benzyl groups are highly solvated in DMF but tend to shrink in THF.79 In conclusion, both DMF and THF are good solvents for PBLG. However, DMF dissolves PBLG better than THF; the interactions between (73) Klee, W. A. Biochemistry 1968, 7, 2731. (74) Quadrifoglio, F.; Urry, D. W. J. Am. Chem. Soc. 1968, 90, 2755. (75) Flory, P. J. Macromolecules 1978, 11, 1138. (76) Schlaad, H.; Smarsly, B.; Below, I. Macromolecules 2006, 19, 4631. (77) Doty, P.; Bradbury, J. H.; Holtzer, A. M. J. Am. Chem. Soc. 1956, 78, 947. (78) Orwoll, R. A. In Polymer Handbook, 3rd ed.; Brandrup, J., Immergut, E. H., Eds.; Wiley-Interscience: New York, 1989. (79) Huang, C.-J.; Chang, F.-C. Macromolecules 2008, 41, 7041.
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Scheme 2. Schematic Representation of the Morphology Transition from Vesicles (a) to Spindles (b) and Then to Spheres (c), as the Degree of Grafting Increases, and from Vesicles (a) to Spindles (d) and Then to Connected-Spindles (e) as fDMF Increases
DMF and PBLG backbone and side chains are relatively stronger. From the extensive experimental results, we learned that both the degree of grafting and the initial solvent nature have marked influence on the self-assembly behavior of the PBLG-g-PEG rod-coil graft copolymers. Shown in Scheme 2 is a schematic illustration of aggregation as functions of the degree of grafting and initial solvent composition. As bearing a lowest degree of grafting, PBLG-g-PEG1 self-assembles into vesicles with THF as initial common solvent. The PBLG backbones should bend in the wall of the vesicle, and the protruded PEG chains stabilize the vesicles in aqueous solution, as illustrated in Scheme 2a. Because of the imperfect helix nature of the PBLG chains, as revealed by CD and 1H NMR analyses (see Figures 9 and 10), the bended state of the PBLG chains in the wall of the vesicles can be achieved without raising the system energy markedly. In addition, as the formed vesicle structure possess very low curvature with smaller interfacial area between PBLG wall and PEG chain with respect to other aggregate morphologies,4-6 the hydrophilic PEG chains can shield the hydrophobic PBLG chains from exposing to water effectively. As a result, stable vesicles are formed. With increasing the degree of grafting, more PEG chains become available to cover PBLG chains; the graft copolymers could form aggregates with relatively larger interfacial area between PEG and PBLG.3 Therefore, vesicles transform to spindles and spheres (spindle has smaller interfacial area than sphere). This progress is schematically represented in Scheme 2a-c. PBLG backbones in the core of the spindles and spheres should align with each other with their direction paralleled with the axial direction of the micelle. Such a kind of rod-chain packing model is evidenced from a recent Brownian dynamics simulations.35 As revealed by the simulations, for the aggregates self-assembled from the rod-coil graft copolymer, the rigid backbones in the core align with each other in a way that the direction of the backbone is perpendicular to the radial direction of the aggregate. In addition, the observed spindle-like shape could be attributed to the dislocation between the parallel polypeptide blocks.52,53 As stated above, DMF is a better solvent for PBLG than THF; when DMF is introduced into the initial solvent, the solubility of the mixed solvent for graft copolymer increases. Therefore, upon the adding of water, the driving force for the aggregation of PBLG chains decreases compared with that using pure THF as initial solvent.3 Under such conditions, the formed aggregates would have a larger interfacial area. In other words, more PBLG chains can be exposed to the surroundings, when DMF is
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introduced. As a result, the vesicles are observed to transform to spindles and connected-spindles with progressively adding DMF into the initial solvent. The structures of the spindles and connected-spindles are schematically represented in parts d and e of Scheme 1, respectively. It is noted that the diameter of the spindles is larger than that of the connected-spindles. That is because when fDMF is relative lower (e.g., fDMF = 0.5), PBLG chains tend to take side-by-side packing, and the dislocation between the paralleled rods is small. Such a packing model can minimize the contact area of hydrophobic PBLG chains with surroundings to lower the free energy. When fDMF is increased, the solubility of solvent becomes better for graft copolymers; more PBLG chains can be exposed to the surroundings. As a result, larger dislocation of PBLG chains, which has larger interfacial area, can be achieved easily, and then connected-spindles with smaller diameters were formed.
Conclusions In summary, we present a first report of the formation of polymeric vesicles from graft copolymer with rigid polypeptide as backbone, and it is also the first example on the morphology transition induced by initial solvent nature for rod-coil type graft copolymer self-assembly system. The effects of the degree of grafting and the nature of initial solvent on self-assembly behavior of PBLG-g-PEG rod-coil graft copolymers in aqueous solution were investigated. With THF as initial solvent, the graft copolymers self-assemble into vesicles, spindle-like micelles, and spherical micelles with increasing of hydrophilic substitution. When DMF is introduced to the initial solvent, the vesicles transform to spindle-like micelles. The spindles transform to connected-spindles as DMF content is further increased. For the graft copolymers with a highest degree of grafting, they form spherical micelles in all the compositions of the mixed initial solvent. These polypeptide-based polymeric aggregates, especially the vesicles, may be a potential candidate for drug carriers and the like. Acknowledgment. This work was supported by National Natural Science Foundation of China (50673026). Support from Projects of Shanghai Municipality (09XD1401400, 082231, 0952nm05100, B502, and 08DZ2230500) is also appreciated. Supporting Information Available: Details regarding the stability testing of the self-assembled aggregates. This material is available free of charge via the Internet at http:// pubs.acs.org.
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