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r-Cyclodextrin-Induced Self-Assembly of a Double-Hydrophilic Block Copolymer in Aqueous Solution Jianhong Liu,‡ Herriyanto R. Sondjaja,† and Kam C. Tam*,†,‡ Singapore-MIT Alliance, and School of Mechanical and Aerospace Engineering, Nanyang Technological UniVersity, 50 Nanyang AVenue, Singapore 639798, Singapore ReceiVed NoVember 18, 2006. In Final Form: February 20, 2007 Double-hydrophilic poly(ethylene oxide)-b-poly(acrylic acid) (PEO-b-PAA) self-assembled into nanostructures in basic solution upon the addition of R-cyclodextrin (R-CD) as a result of the complexation between R-CD and PEO. The nanostructures produced were spherical in shape as observed by transmission electron microscopy (TEM) and possessed radii that were much larger than that of a single stretched polymeric chain. The ratio of Rg/Rh (where Rg is the z-average radius of gyration and Rh is the hydrodynamic radius) obtained from laser light scattering (LLS) was approximately ∼1.0, and the aggregation number was ∼4100. The zeta-potential of complex particle was -45 mV, suggesting that the particle possessed a stable negatively charged surface, attributed to ionized PAA segments. The above results suggested that the nanostructures formed in the PEO-b-PAA/R-CD solution at high pH were likely to be spherical vesicles.
Introduction Recently, increasing attention has been paid to polymer inclusion complexes (PICs) formed between various polymers and cyclodextrins (CDs). Such PICs are based on noncovalent host-guest interactions and are useful building blocks for constructing supramolecular structures.1-3 Cyclodextrins are cyclic molecules that consist of six (R-), seven (β-), or eight (γ-) glucose units linked through R-1-4-glycosidic linkages. They are readily soluble in water and are capable of selective inclusion of a wide range of guest molecules.4 The first PIC was reported by Harada and co-workers in 1990. When poly(ethylene glycol) was added to a saturated R-CD solution, white crystalline compounds were obtained in high yield.5 Following this, a series of studies on this topic have been reported by Harada’s group covering hydrophilic and hydrophobic polymers,6-10 and inorganic polymers.11 This study was then extended to biodegradable polymers12,13 and block copolymers.14-16 A pH-dependent * To whom correspondence should be addressed. Fax: (65) 6791 1859. E-mail:
[email protected]. † Singapore-MIT Alliance. ‡ School of Mechanical and Aerospace Engineering. (1) Harada, A. Coord. Chem. ReV. 1996, 148, 115-133. (2) Herrmann, W.; Keller, B.; Wenz, G. Macromolecules 1997, 30, 49664972. (3) Harada, A.; Okada, M.; Kawaguchi, Y.; Kamachi, M. Polym. AdV. Technol. 1999, 10, 3-12. (4) Wenz, G. Angew. Chem., Int. Ed. Engl. 1994, 33, 803-822. (5) Harada, A.; Kamachi, M. Macromolecules 1990, 23, 2821-2823. (6) Harada, A.; Li, J.; Kamachi, M. Nature 1992, 356, 325-327. (7) Harada, A.; Li, J.; Kamachi, M. Macromolecules 1994, 27, 4538-4543. (8) Harada, A.; Okada, M.; Kamachi, M. Acta Polym. 1995, 46, 453-457. (9) Harada, A.; Nishiyama, T.; Kawaguchi, Y.; Okada, M.; Kamachi, M. Macromolecules 1997, 30, 7115-7118. (10) (a) Okumura, H.; Okada, M.; Kawauchi, Y.; Harada, A. Macromolecules 2000, 33, 4297-4298. (b) Harada, A.; Kataoka, K. Prog. Polym. Sci. 2006, 41, 949-982. (11) Okumura, H.; Okada, M.; Kawaguchi, Y.; Harada, A. Macromolecules 2000, 33, 4297-4298. (12) Harada, A.; Kawaguchi, Y.; Nishiyama, T.; Kamachi, M. Macromol. Rapid Commun. 1997, 18, 535-539. (13) Huh, K. M.; Ooya, T.; Sasaki, S.; Yui, N. Macromolecules 2001, 34, 2402-2404. (14) Lee, S. C.; Choi, H. S.; Ooya, T.; Yui, N. Macromolecules 2004, 37, 7464-7468. (15) Choi, H. S.; Lee, S. C.; Yamamoto, K.; Yui, N. Macromolecules 2005, 38, 9878-9881. (16) Choi, H. S.; Ooya, T.; Yui, N. Macromol. Biosci. 2006, 6, 420-424.
polypseudorotaxane obtained from R-CD and a triblock copolymer (PEI-b-PEG-b-PEI) was reported by Yui et al., where the CD-triblock polymer complexes were synthesized in both organic and aqueous solutions and the solid properties of the PIC were investigated by NMR and XRD techniques.16-18 Tonelli and co-workers have investigated the unique properties of PICs in confined geometry, where narrow channels were produced in crystalline matrices of host clathrates.17,18 Although a wide range of polymers have been investigated with various cyclodextrins, these studies mainly focused on the PIC preparation techniques and the characterization of solid phases. The solution properties, such as the self-assembly behavior, dissociation, particle size, and morphology, were not commonly reported. These solution properties, especially the assembly behavior, are vital for the potential applications of such systems in biomedical science, such as in controlled drug delivery. Vesicles represent a class of microstructures commonly found in many biological systems. These structures possess unique characteristics, and synthetic variants have been prepared from amphiphilic block copolymers. The physicochemical properties and their applications as delivery vehicles for drugs and biomolecules have been reported by several research groups.19 We report here a PIC produced from R-CD and a doublehydrophilic diblock copolymer, poly(ethylene oxide)-b-poly(acrylic acid) (PEO-b-PAA), which focused on the solution property and the mechanism driving the formation of the PIC. Complexes produced from a diblock copolymer and cyclodextrin were previously reported by Yui et al.,20 but their PIC product (CD and PEG grafted dextrans) was gel-like. The PIC reported here consists of pH-responsive vesicular structures dispersed in an aqueous solution. The potential advantage of the vesicle (17) Rusa, C.; Tonelli, A. Macromolecules 2000, 33, 1813-1818. (18) Rusa, C.; Tonelli, A. Macromolecules 2000, 33, 5321-5324. (19) (a) Azzam, T.; Eisenberg, A. Angew, Chem., Int. Ed. 2006, 45, 74437447. (b) Choucair, A.; Lavigueur, C.; Eisenberg, A. Langmuir 2004, 20, 38943900. (c) Soo, P. L.; Eisenberg, A. J. Polym. Sci., Part B: Polym. Phys. 2004, 42, 923-938. (d) Bae, Y.; Fukushima, S.; Harada, A.; Kataoka, K. Angew. Chem., Int. Ed. 2003, 42, 4640-4643. (e) Kataoka, K.; Harada, A.; Nagasaki, Y. AdV. Drug DeliVery ReV. 2001, 47, 113-131. (20) Huh, K. M.; Ooya, T.; Lee, W. K.; Sasaki, S.; Kwon, I. C.; Jeong, S. Y.; Yui, N. Macromolecules 2001, 34, 8657-8662.
10.1021/la063365r CCC: $37.00 © 2007 American Chemical Society Published on Web 03/29/2007
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Figure 1. PEO-b-PAA/R-CD complex obtained at pH 3 (turbid solution) and pH 10 (clear solution).
reported here over previous systems lies in the possibility that the vesicular structure of the PIC produced from R-CD and PEOb-PAA may be controlled by external stimuli such as pH or by the introduction of host molecules. Experimental Methods The block copolymer PEO45-b-PAA113 (Mw 1.18 × 104) used in this study was synthesized by atom transfer radical polymerization technique.21 For the study of CD-polymer complexes, PEO-b-PAA solution of 0.1 wt % was first prepared by dissolving 2 mg of the polymer in deionized (DI) water. 145 mg of R-CD (0.15mmol) was added to 1 mL of the above solution at pH of 3 and 10, respectively. The resultant solutions were vigorously stirred and agitated ultrasonically to promote dissolution, and they were stored overnight at room temperature prior to use. The microstructure of the sample solution prepared at pH 10 was examined using a Brookhaven Laser Light Scattering system, which consists of a BI-200SM goniometer and a BI-9000AT digital correlator. A 488 nm vertically polarized argon ion laser was used as the light source. The zeta-potential of the solution was measured using a Brookhaven Zeta-Potential Analyzer. The calorimetric measurements were carried out using the Microcal isothermal titration calorimeter (ITC). It has a reference and a sample cell of approximately 1.35 mL, and both cells are insulated by an adiabatic shield. The titration was carried out at 25.0 ( 0.02 °C. The microscopic property of the solution was studied using a JEOL TEM-2010 transmission electron microscope operated at 160 kV. The sample was prepared on a copper grid precoated with carbon and stained with cesium hydroxide (CsOH).
Results and Discussion The two sample solutions prepared consisted of the same amount of PEO-b-PAA and R-CD, but one was at pH 3 and the other at pH 10. After equilibration for 12 h at room temperature, the solution at low pH of 3 became turbid with white precipitates, while the solution at high pH of 10 remained clear as shown in Figure 1. The precipitation was considered to be evidence of complex formation between PEO-b-PAA and R-CD. The pHsensitivity of the resultant complex was attributed to the presence of PAA segment. As confirmed by Harada and based on molecular model calculation,5 the PEO block can be encapsulated by R-CD (with a cavity diameter of 5.7 Å). However, PAA chain cannot be encapsulated due to steric hindrance of carboxylic groups. During the inclusion complexation, the PEO block became hydrophobic due to the formation of intermolecular hydrogen bonding among the R-CDs threaded along the polymer chain.20 At pH 3, the PAA segments were less hydrophilic as a result of (21) Matyjaszewski, K.; Xia, J. Chem. ReV. 2001, 101, 2921-2990.
Figure 2. Calorimetric titration of 0.15 mol/L R-CD into 0.1 wt % solution of PEO-b-PAA at pH 10: (a) thermogram showing cell feedback versus time; (b) differential enthalpy curve versus the R-CD concentration.
the protonation of carboxylic groups, which caused the precipitation of CD-polymer complexes, while at pH 10, the ionization of carboxylic groups to COO- induced significant hydrophilic character to the PAA segments, resulting in the complete dissolution of the PEO-b-PAA/R-CD complex. The complexation between PEO-b-PAA and R-CD was also confirmed by isothermal titration calorimetric (ITC) study. A saturated R-CD solution (0.15 M, pH 10) was titrated into 0.1 wt % PEO-b-PAA solution at pH 10. The cell feedback signal (CFB) during the titration process is shown in Figure 2a. Integration of the area of CFB gave the differential enthalpy curve (Figure 2b). As the titration of R-CD to water produced negligible heat change,22 the enthalpy curve in Figure 2b can only be ascribed to the complexation between R-CD and the polymer (PEO segments). The binding process was exothermic, indicating that the complex formation between PEO-b-PAA and R-CD was energetically favorable. The binding took place at the first injection of R-CD solution. Upon further addition of R-CD, the enthalpy curve progressively approached the baseline, indicating the completion of binding between R-CD and PEOb-PAA. Analysis of the data confirmed that one R-CD was bound to 2 repeat units of EO segments. The clear solution of PEO-b-PAA/R-CD complex at pH 10 was examined by dynamic light scattering (DLS). Figure 3 shows the distribution functions at different scattering angles from 60° to 135° obtained from DLS measurments. Monomodal relaxation distributions were observed, indicating that only one type of particle was present in solution. The dependence of the decay rate Γ on the square of the scattering vector, q2, is shown in Figure 4. q is defined as q ) 4πn sin(θ/2)/λ, where n is the refractive index of solvent, θ is the scattering angle, and λ is the wavelength of the incident laser light in a vacuum. A linear relationship between Γ and q2 confirmed the distribution functions in Figure 3 were caused by translational diffusion of the particles. Rh of PEO-b-PAA/R-CD complex was determined to be 165 nm using the Stoke-Einstein equation. The Rg and the apparent weight-average molar mass (Mwapp) determined from static light scattering (SLS) were 159 nm and (22) Zheng, P. J.; Wang, C.; Hu, X.; Tam, K. C.; Li, L. Macromolecules 2005, 38, 2859-2864.
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Figure 5. TEM micrograph of nanoparticles in 0.1 wt % PEO-bPAA solution with 0.15 M R-CD at pH 10. Scheme 1. Self-Assembly of PEO-b-PAA into Vesicular Nanostructures at pH 10 Induced by the Complexation between PEO Segments and r-CD Figure 3. Relaxation distribution functions obtained from DLS measurement of PEO-b-PAA solution in the presence of R-CD at pH 10.
Figure 4. The dependence of the decay rate Γ on the square of scattering vector q.
1.4 × 108, respectively. The aggregation number of the PEOb-PAA/R-CD complex was evaluated from Mwapp/Mwc, where Mwc was the molar mass of one PEO-b-PAA/R-CD complex assuming that all of the ethylene oxide (EO) units in PEO block were encapsulated in R-CD cavities with a 2:1 ratio (2 EO:1 R-CD).3,5,8 The aggregation number of the nanoparticles formed by PEO-b-PAA/R-CD complexes was ∼4100 and would be larger when the binding ratio between EO and R-CD was less than the optimum of 2:1. The zeta-potential of the PEO-b-PAA/R-CD (0.1 wt % polymer and 0.15 M R-CD) solution at pH 10 was -45 mV. As the PAA block was ionized into COO- groups at this pH, the zeta-potential value strongly suggested that the surface of the PEO-b-PAA/R-CD aggregates consisted of ionized PAA segments. The morphology of the PEO-b-PAA/R-CD nanoparticles was studied by TEM. Figure 5 shows the TEM micrograph of the 0.1 wt % PEO-b-PAA solution in 0.15 M R-CD at pH 10. Vesicular structures were observed, and they represented the complex formed by R-CD and PEO-b-PAA. With PEO block encapsulated by R-CDs, neighboring PEO-b-PAA/R-CD complex molecules would form intermolecular hydrogen bonds between R-CDs threaded along PEO chains (Scheme 1c).20 These hydrogen-
bonded R-CD-PEO complexes self-aggregate into the most stable structure, and under this condition a vesicular structure was produced (Scheme 1d). For the PEO-b-PAA/R-CD complex in pH 10 solution, the parameter F (ratio Rg/Rh) commonly used to examine the morphology of the microstructure of aggregates23,24 was found to be ∼0.96, which was close to 1.0. This value differed from the expected theoretical values for a uniform sphere (0.775) or a Gaussian chain (1.50), but was very close to the expected value of 1.0 for a vesicle. As compared to the length of one single PEO-b-PAA molecular chain (approximately 51 nm when stretched), the measured radius (165 nm) also suggested a vesicular morphology because this value was much larger than the radius of a core-shell micellar structure. For a fully stretched chain representing a shell thickness of 51 nm, F was calculated to be ∼0.87. Hence, the measured F of 0.96, which corresponds to a shell thickness of ∼14 nm, may indicate that the chain is in a more random configuration. Considering the Mw of the aggregate (1.4 × 108), aggregation number (4.1 × 103), and the morphology as derived from TEM, it is most likely that a vesicular structure was produced by R-CD-induced micellization of PEOb-PAA at high pH, which is comprised of a R-CD/PEO shell with ionized PAA chains extended on the external and internal regions of the vesicle as shown in Scheme 1. (23) Burchard, W. AdV. Polym. Sci. 1983, 48, 1-124. (24) Ravi, P.; Wang, C.; Tam, K. C.; Gan, L. H. Macromolecules 2003, 36, 173-179.
Self-Assembly of a Double-Hydrophilic Block Copolymer
Conclusions We report for the first time a CD-induced assembly of a double hydrophilic block copolymer PEO-b-PAA to produce a vesicular nanostructure that has potential application as a drug delivery vehicle. The Rh and Rg values of the nanostructure obtained from LLS measurement were 165 and 159 nm, respectively, with a zeta-potential of -45 mV. The apparent molar mass was determined to be 1.4 × 108, and the aggregation number was 4.1 × 103. All of the above results were obtained in aqueous solution.
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Acknowledgment. We acknowledge the financial support provided by the Singapore-MIT Alliance and the Ministry of Education, Singapore. Supporting Information Available: Atom transfer radical polymerization and characterization of the block copolymer, PEO-bPAA. This material is available free of charge via the Internet at http://pubs.acs.org. LA063365R
