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Formation of Internally Nanostructured Triblock Copolymer Particles Karin Bryskhe,*,† Karin Schille´n,† Ulf Olsson,† Anan Yaghmur,‡ and Otto Glatter‡ Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden, and Physical Chemistry, Institute of Chemistry, University of Graz, Graz, Austria Received April 29, 2005. In Final Form: June 30, 2005 Particles with an internal structure have been found in dilute water solutions of a triblock copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), which has short hydrophilic PEO endblocks compared to the central hydrophobic PPO block (EO5PO68EO5, L121). The properties of the block copolymer particles (i.e., their structure, size, and time stability) have been investigated using cryogenic transmission electron microscopy (cryo-TEM) in combination with dynamic light scattering (DLS) and turbidity measurements. The particles were formed in dilute solutions by quenching the temperature to temperatures where the reversed hexagonal phase is in equilibrium with a solution of unaggregated L121 copolymers (L1). From the DLS measurements, a mean hydrodynamic radius of 158 nm was extracted. The time-scan turbidity measurements were found to be unchanged for about 46 h. At higher copolymer concentrations, a reversed hexagonal phase (H2) exists in the L121/water system. SAXS was used to investigate the internal structure of the dispersed L121-based particles containing 15 wt % L121. It was found that the internal structure transforms from H2 to an inverse micellar system (L2) as the temperature increases from 37 to 70 °C.
Introduction One class of water-soluble block copolymers that has attracted great interest in the literature is the triblock copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), often abbreviated as PEO-PPOPEO or EOa-POb-EOa. These copolymers exist in a wide range of different compositions and display rich phase behavior in water depending on their relative block lengths.1,2 In dilute aqueous solution, the more hydrophilic copolymers (i.e., those with long PEO blocks) self-assemble into micelles with a PPO core and a PEO corona.3,4 The properties and shape of these micelles have been characterized carefully using various scattering techniques and theoretical model calculations; see, for example, refs 5-8. However, hollow structures (i.e., vesicles) have also recently been discovered to exist in an aqueous system of one of the most hydrophobic PEO-PPO-PEO copolymers, EO5PO68EO5 (L121).9,10 The present work focuses on the investigation of dilute aqueous solutions of L121, where particles with reversed hexagonal structure have been found under certain conditions and visualized by the cryogenic transmission † ‡
Lund University. University of Graz.
(1) Chu, B.; Zhou, Z. In Nonionic Surfactants: Polyoxyalkylene Block Copolymers; Nace, V. M., Ed.; Marcel Dekker: New York, 1996; Vol. 60, p 67. (2) Alexandridis, P. Curr. Opin. Colloid Interface Sci. 1997, 2, 478. (3) Tuzar, Z.; Kratochvı´l, P. In Surface Colloid Science; Matijevic´, E., Ed.; Plenum Press: New York, 1993; Vol. 15, p 1. (4) Almgren, M.; Brown, W.; Hvidt, S. Colloid Polym. Sci. 1995, 273, 2. (5) Nolan, S. L.; Phillips, R. J.; Cotts, P. M.; Dungan, S. R. J. Colloid Interface Sci. 1997, 191, 391. (6) Mortensen, K.; Brown, W. Macromolecules 1993, 26, 4128. (7) Linse, P. J. Phys. Chem. 1993, 97, 13896. (8) Schille´n, K.; Brown, W.; Johnsen, R. M. Macromolecules 1994, 27, 4825. (9) Schille´n, K.; Bryskhe, K.; Mel’nikova, Y. S. Macromolecules 1999, 32, 6885. (10) Bryskhe, K.; Jansson, J.; Topgaard, D.; Schille´n, K.; Olsson, U. J. Phys. Chem. B 2004, 108, 9710.
electron microscopy (cryo-TEM) technique. Several review articles where the dispersed cubic phases (“cubosomes”)11 and dispersed reversed hexagonal phases (“hexosomes”)12 in aqueous systems of lipids, polymers, and surfactant is discussed can be found in the literature.13-16 The suggested biological relevance11,17 of structures with reversed curvature, such as reversed hexagonal and cubic phases, has led to a number of publications on the subject.18-23 A block copolymer of the same type as that investigated in this study (F127) or poloxamer 407 (EO97PO68EO97) has been used to stabilize hexosomes and cubosomes.12,18,24-28 Only one earlier study has been found where nanostructured particles are formed by the block copolymer itself.29 Other dispersion agents such as bile (11) Larsson, K. J. Phys. Chem. 1989, 93, 7304. (12) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1997, 13, 6964. (13) Almgren, M.; Edwards, K.; Gustafsson, J. Curr. Opin. Colloid Interface Sci. 1996, 1, 270. (14) Larsson, K. Curr. Opin. Colloid Interface Sci. 2000, 5, 64. (15) Almgren, M.; Edwards, K.; Karlsson, G. Colloids Surf., A 2000, 174, 3. (16) Almgren, M. Aust. J. Chem. 2003, 56, 959. (17) Landh, T. Cubic Cell Membranes Architectures. Lund University, 1996. (18) Nakano, M.; Teshigawara, T.; Sugita, A.; Leesajakul, W.; Taniguchi, A.; Kamo, T.; Matsuoka, H.; Handa, T. Langmuir 2002, 18, 9283. (19) Yang, D.; O’Brien, D. F.; Marder, S. R. J. Am. Chem. Soc. 2002, 124, 13388. (20) Borne, J.; Nylander, T.; Khan, A. J. Phys. Chem. B 2002, 106, 10492. (21) Zheng, L. Q.; Um, J. Y.; Chung, H. S.; Kwon, I. C.; Li, G. Z.; Jeong, S. Y. J. Dispersion Sci. Technol. 2003, 24, 123. (22) Boyd, B. J. Int. J. Pharm. 2003, 260, 239. (23) Lynch, M. L.; Ofori-Boateng, A.; Hippe, A.; Kochvar, K.; Spicer, P. T. J. Colloid Interface Sci. 2003, 260, 404. (24) Landh, T. J. Phys. Chem. 1994, 98, 8453. (25) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1996, 12, 4611. (26) Leesajakul, W.; Nakano, M.; Taniguchi, A.; Handa, T. Colloids Surf., B 2004, 34, 253. (27) Kamo, T.; Nakano, M.; Leesajakul, W.; Sugita, A.; Matsuoka, H.; Handa, T. Langmuir 2003, 19, 9191. (28) Siekmann, B.; Bunjes, H.; Koch, M. M. H.; Westesen, K. Int. J. Pharm. 2002, 244, 33.
10.1021/la051157d CCC: $30.25 © 2005 American Chemical Society Published on Web 08/09/2005
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Imaging by Cryo-TEM
Figure 1. Phase diagram of the EO5PO68EO5/D2O system (adapted from ref 36). The boundaries of the various one-phase regions are indicated with solid lines. The three-phase lines are marked with bold broken lines. L1, LR, H2, and L2 correspond to isotropic polymer solution, lamellar phase, reversed hexagonal phase, and concentrated polymer solution, respectively.
salts and caseins have also been studied.30-32 Cubosomes and hexosomes produced in the monoglyceride/water system and stabilized by F127 have been studied with a number of techniques. The internal structure has been studied by SAXS and 13C NMR,33 and the particles have been imaged by atomic force microscopy;34 the same particles were found to take up salt added to the dispersions.33 A reversible phase transition of the internal structure of particles formed in the monoglyceride/F127/ water system is observed when the temperature is cycled.35 This indicates that the internal structure of the particles is an equilibrium structure. The PEO-PPO-PEO triblock copolymer used in this study, denoted L121, was a gift from BASF Corporation and used without further purification. It has a nominal molecular weight of 4400 g/mol, and the average composition is EO5PO68EO5. At low concentrations and temperatures, the L121/water system displays an isotropic L1 phase with the copolymer present mostly as unimers (individual copolymer chains), with a hydrodynamic radius (RH) of 1.9 nm at 10 °C.9,10 The L121 copolymers do not self-assemble into micelles in the L1 phase. The binary L121/H2O and L121/D2O phase diagrams were published earlier by our group.9,10,36 In those studies, it was also found that L121 vesicles can be formed in the L1 + LR two-phase region either by extrusion9 or by simply heating the sample from the L1 phase into the L1 + LR two-phase region.10 Sample Preparation The nanostructured particles in this study are prepared by first heating the L121 solutions (c ) 0.001, 0.01, 0.05, 0.6, and 15 wt %) to a temperature of 80 °C, where the sample was stirred and where an L121-in-water emulsion (L2 + L1) is formed (Figure 1). Thereafter, the temperature of the solutions was quickly lowered to either 35 or 40 °C. In this step, the emulsion is brought (29) Oh, K. T.; Bronich, T. K.; Kabanov, A. V. J. Controlled Release 2004, 94, 411. (30) Lindstro¨m, M.; Ljusberg-Wahren, H.; Larsson, K.; Borgstro¨m, B. Lipids 1981, 16, 749. (31) Bucheim, W.; Larsson, K. J. Colloid Interface Sci. 1987, 117, 582. (32) Gustafsson, J.; Nylander, T.; Almgren, M.; Ljusberg-Wahren, H. J. Colloid Interface Sci. 1999, 211, 326. (33) Nakano, M.; Sugita, A.; Matsuoka, H.; Handa, T. Langmuir 2001, 17, 3917. (34) Neto, C.; Aloisi, G.; Baglioni, P.; Larsson, K. J. Phys. Chem. B 1999, 103, 3896. (35) de Campo, L.; Yaghmur, A.; Sagalowicz, L.; Leser, M. E.; Watzke, H.; Glatter, O. Langmuir 2004, 20, 5254. (36) Bryskhe, K.; Schille´n, K.; Lo¨froth, J.-E.; Olsson, U. Phys. Chem. Chem. Phys 2001, 3, 1303.
The particles formed were first imaged by cryo-TEM, a technique that has been described elsewhere,37 and the micrographs are presented in Figure 2. In these images, it is seen that these particles have an internal structure. However, the structure cannot be recognized as having hexagonal symmetry, which is the predicted structure. The particles are, as mentioned before, formed in the two-phase region of L1 + H2 (Figure 1); therefore, a reversed hexagonal structure is expected. The H2 phase present at higher concentrations was previously identified using both SAXS and NMR techniques, and its hexagonal lattice parameters were determined.36 In all particles imaged, parallel lines near the surface of the particles are observed. The dark and light lines observed in the images are regions in the particles of higher or lower electron density, respectively. The repeat distance of the lines was determined to 14 nm using the image software of the microscope, which is of the same order as the nearest-neighbor distances in the concentrated lamellar and hexagonal structures in the same system.36 The observation that the parallel lines are disappearing by moving inside the particles indicates that the structure becomes more random, which could be an effect of the increase of the thickness of the particle approaching the center of the particle. The almost spherical shape of the particles indicates significant surface tension.
Investigation of the Internal Structure by SAXS The internal structure of the particles was further examined by small-angle X-ray scattering (SAXS). The SAXS measurements were performed on 15 wt % L121 solution at 37-70 °C, scanning the different two-phase regions (Figure 1). Here, a higher concentration was needed for a better scattering signal. The SAXS measurements were performed at 37-70 °C. The SAXS equipment that was used consisted of a SAXSess camera38 (AntonPaar, Graz, Austria), which is connected to an X-ray generator (Philips, PW 1730/10) operating at 40 kV and 50 mA with a sealed-tube Cu anode. A Go¨bel mirror is used to convert the divergent polychromatic X-ray beam into a focused line-shaped beam of Cu KR radiation (λ ) 0.154 nm). The 2D scattering pattern is recorded by an imaging-plate detector (model Fuji BAS1800 from Raytest, Straubenhardt, Germany) and integrated to obtain the 1D scattering function I(q) using SAXSQuant software (Anton Paar, Graz, Austria), where q is the length of the scattering vector, defined by q ) (4π/λ)sin θ/2, with λ being the wavelength and θ being the scattering angle. The interplanar distances d between two reflecting planes is given by d ) 2π/q, which enables us to calculate the corresponding mean lattice parameter a. For hexagonal liquid-crystalline phases, the reflection law is given as follows: (3a/4d)2 ) 1, 3, 4, 7,... The scattering profiles of the so-called L2 phase show only one broad correlation peak, for which the position of the observed maximum is shifted to lower q values because of the “smearing effects” of the line-shaped primary beam. These profiles were desmeared by fitting these data with the generalized indirect fourier transformation (GIFT) method.39 For the L2 phase, d is instead the characteristic distance, which may be interpreted as the distance between the water droplets in the polymer phase. The sample, a solution of 15 wt % L121 in water, was placed at room temperature into the sample holder (capillary in a metal block, temperature controlled by a Peltier element, (0.1 °C) and equilibrated at each experimental temperature for at least 10 min before measurement. All temperature scans were performed in the heating direction, and the measuring time consisted of 45 min of exposure time taken for the sample. (37) Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Talmon, Y. J. Electron Microsc. Tech. 1988, 10, 87. (38) Bergmann, A.; Orthaber, D.; Scherf, G.; Glatter, O. J. Appl. Crystallogr. 2000, 33, 869.
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Figure 2. Cryo-TEM images of nanostructured block copolymer particles formed in an aqueous solution of 0.6 wt % L121 prepared by quenching the temperature from 80 to 35 °C.
Figure 4. Turbidity vs time for a nanostructured particle solution of 0.6 wt % L121. O and b indicate two separate experiments.
Figure 3. Experimental small-angle X-ray scattering curves after the subtraction of water scattering for the block copolymer aqueous dispersion containing 15 wt % L121. The temperaturedependence measurements of the scattering curves was carried out in the range of 37-70 °C. The curves are shifted by a constant offset factor for better visibility. The black arrows indicate the position of the observed peaks.
turbidity measurements on a 0.6 wt % L121 particle solution as a function of time are displayed in Figure 4. The turbidity remains constant for approximately 46 h, and after that, a small decrease in turbidity is observed. This is possibly due to slow sedimentation of the particles because this sample finally macroscopically phase separates, which can be observed visually. Increased stability at lower concentrations is therefore anticipated.
Particle Sizing by DLS A selection of the smeared experimental SAXS curves (X-ray scattering intensity vs the magnitude of the scattering vector) are presented in Figure 3. From the SAXS curves in Figure 3 and the phase diagram in Figure 1, we may conclude that the interior of the particles has a reversed hexagonal structure at 37 and 45 °C. The mean lattice parameter of the reversed hexagonal structure of the particles formed at 15 wt % block copolymer and 45 °C is determined to 22.6 nm, which is comparable to 20.7 nm36 in the concentrated reversed hexagonal phase of 60 wt % at 40 °C. The difference in the lattice parameter between the dispersed and the nondispersed bulk sample is attributed to the fact that in the one-phase region, at a constant temperature, the lattice parameter of the pure phases increases with sample hydration and thus it is lower than that of the dispersed phase. However, in the two-phase region, the fully hydrated phases coexist in equilibrium with excess water; therefore, the lattice parameter is constant with increasing water content, and its value is close to that for the corresponding dispersion. In the two-phase region of L2 + L1 at temperatures above 45 °C, the characteristic distance for the L2 phase was changing from 16.3 to 13.0 nm as the temperature was varied from 50 to 70 °C. Therefore, the nanostructures become smaller because of the fact that increasing temperature leads to a decrease in the water solubilization capacity. This result confirms recent investigations34 done on both dispersed and nondispersed bulk samples based on monolinolein (MLO, unsaturated monoglyceride).
Stability Test The L121 nanostructured particles studied here are not stabilized with any additional stabilizer. Their time stability at 35 °C was investigated by turbidity (τ) measurements performed on a Perkin-Elmer Lambda 14 UV-vis double-beam spectrophotometer at a wavelength of 532 nm with simultaneous subtraction of the background using pure water. The two separate
The L121 particles were also studied at short times by dynamic light scattering measurements. The measurements were carried out using an ALV/DLS/SLS-5000F, CGS-8F-based compact goniometer system from ALV Gmbh (Langen, Germany) and a CW diode-pumped solid-state Nd:YAG Compass-DPSS laser (λ ) 532 nm) from Coherent (Santa Barbara, CA). Two multipledelay-time digital correlators (ALV-5000/E and ALV-5000/FAST) with a total of 320 exponentially spaced channels were employed to construct the normalized time-correlation function of the scattered intensity. The experimental setup, with the difference being that the refractive index matching liquid used here was decalin instead of toluene, and DLS data analysis have been described elsewhere. 40 The z-averaged apparent hydrodynamic radius (RH,app) was measured as a function of low L121 concentrations at 35 °C (Figure 5 a). Each RH,app value was obtained from the collective diffusion coefficient extrapolated to q ) 0 (Dq)0) using RH,app ) kT/(6πη0Dq)0), where k is the Boltzmann constant, T is the temperature, η0 is the viscosity of the solvent, and Dq)0 is the collective diffusion coefficient at zero angle. For the low concentrations used here, the effect of interactions can be neglected. The hydrodynamic radius at 35 °C is approximately 150 nm for all concentrations investigated. The temperature dependence of RH,app for the particles at two different concentrations, 0.01 and 0.05 wt %, was also investigated (Figure 5b). The particles are polydisperse in size (and shape), which is observed in the cryo-TEM images. The micrographs shown here (in Figure 2) display very large particles. In the DLS measurements performed, the diffusion coefficient show a q dependence. This indicates that different sizes contribute differently to the mean according to the angular dependence of their form factor.41 (39) Bergmann, A.; Fritz, G.; Glatter, O. J. Appl. Crystallogr. 2000, 33, 1212. (40) Jansson, J.; Schille´n, K.; Olofsson, G.; da Silva, R. C.; Loh, W. J. Phys. Chem. B 2004, 108, 82.
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Figure 5. (A) Apparent hydrodynamic radius (RH,app) of the particles as a function of L121 concentration at 35 °C. (B) Apparent hydrodynamic radius (RH,app) of the particles at two temperatures and two concentrations. O ) 0.05 wt % L121, and b ) 0.01 wt % L121. However, this effect is not very pronounced for particles smaller than the wavelength of the light. The general trend is that the size of the particles is smaller at 40 °C compared to that at 35 °C. Because the same behavior is found at different concentrations, it could be an indication that the temperature to which the L1 + L2 solutions (at 80 °C) are quenched or the rate of temperature change affects the final size of the particle. When the emulsion of block copolymer in water is brought into the two-phase region (L1 + H2) and a reversed hexagonal structure is formed from block copolymer emulsion droplets, it is reasonable to assume that if no aggregation of the droplets occur then the size of the particles will depend on the size of the emulsion droplets when the emulsion enters the two-phase region of L1 + H2. From the phase diagram in Figure 1, this temperature can be determined to be 46 °C.
Conclusions We have investigated dilute aqueous solutions of a block copolymer, L121 having the nominal composition of EO5PO68EO5. Nanostructured particles are formed without any stabilizer simply by changing the temperature of a (41) Schurtenberger, P.; Newman, M. E. In Environmental Particles; Buffle, J., van Leeuwen, H. P., Eds.; Lewis Publishers: Boca Raton, FL, 1993.
polymer-in-water emulsion. From DLS measurements, the particle mean hydrodynamic radius was determined to 150 nm. Their time stability at 35 °C was investigated, and it was found that the turbidity remains constant for approximately 46 h. Cryo-TEM imaging shows that the particles have an internal structure. From SAXS measurements, we may conclude that the interior of the particles has reversed hexagonal structure at 37 and 45 °C. It is reasonable to assume that the particle size will depend on the size of the emulsion droplets. However, further investigations of the time and temperature effects on the size of the emulsion droplets are needed to confirm this hypothesis. Additional studies on the time dependence of the internal structure of the nanostuctured particles would also be of great interest. Acknowledgment. We are grateful to Gunnel Karlsson for the cryo-TEM imaging and Håkan Wennerstro¨m and Markus Johnsson for fruitful discussions. This work was supported by the Swedish Research Council (VR) and the Center for Amphiphilic Polymers from Renewable Resources (CAP). LA051157D
