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Articles Spontaneous Generation of Multilamellar Vesicles from Ethylene Oxide/Butylene Oxide Diblock Copolymers J. Keith Harris,*,†,‡ Gene D. Rose,‡ and Merlin L. Bruening† Chemistry Department, Michigan State University, East Lansing, Michigan 48824, and The Dow Chemical Company, Corporate Research and Development, Midland, Michigan 48674 Received December 6, 2001. In Final Form: April 5, 2002 Vesicular structures are promising materials for encapsulation applications, and vesicles composed from block copolymers are attractive in this regard because of their stability and tunability. However, vesicle formation from copolymers generally requires specialized procedures. This article demonstrates that ethylene oxide/butylene oxide diblock copolymers spontaneously form micrometer-sized, multilamellar vesicles (“onions”) over a broad range of concentrations upon simple mixing with water. These structures likely form due to the relatively large volume-to-length ratio of the butylene oxide hydrophobe, and vesicle formation also depends on the length of the ethylene oxide headgroup. Structures were characterized using plane-polarized light microscopy, dynamic light scattering, and cryogenic scanning electron microscopy. The multilamellar vesicles survive sonication and moderate shear. Vesicle size can be reduced by extrusion through a porous membrane, but final diameter is independent of shear rate and membrane pore size.
Introduction Multilamellar vesicles (Figure 1) show great potential as controlled-release materials for water-insoluble pharmaceutical, agricultural, household, and personal care products.1-3 Compared to unilamellar vesicles, these structures have a much larger hydrophobic volume for encapsulating hydrophobic agents. Although spontaneous formation of multilamellar vesicles (MLVs) from phospholipids is common, lipids possess inherent chemical and mechanical limitations.4 Amphiphilic diblock copolymers offer several advantages for vesicle formation relative to such conventional surfactants. Nonionic copolymers demonstrate lower pH and saline sensitivity than ionic surfactants, and multilamellar vesicles produced from these copolymers would be expected to show this same reduced sensitivity.5 Additionally, ethoxylated materials have inherent biocompatiblilty, making them appealing as drug delivery agents.6 The advantage that copolymers offer which is of most interest to this work, however, is the ability to produce surfactants with various geometric configurations by careful selection of monomer type and block length. Recent studies demonstrate the utility of diblock copolymers in micellar structure formation. Laibin and Eisenberg showed that polystyrene/poly(acrylic acid) * To whom correspondence should be addressed. E-mail:
[email protected]. † Michigan State University. ‡ The Dow Chemical Company. (1) Lasic, D. D. In Vesicles; Rosoff, M., Ed.; Marcel Dekker: New York, 1996; p 448. (2) Allen, C.; Maysinger, D.; Eisenberg, A. Colloids Surf., B 1999, 16, 3. (3) Ding, J.; Liu, G. J. Phys. Chem. B 1998, 102, 6107. (4) Gerasimov, O. V.; Rui, Y.; Thompson, D. H. In Vesicles; Rosoff, M., Ed.; Marcel Dekker: New York, 1996; p 682. (5) Rosen, M. J. Surfactants and Interfacial Phenomena; WileyInterscience: New York, 1989; pp 21-23. (6) Watson, K. J.; Anderson, D. R.; Nguyen, S. T. Macromolecules 2001, 34, 3507.
Figure 1. Schematic diagram of unilamellar (left) and multilamellar vesicles (MLVs).
diblock compositions form a variety of structures, including vesicles.7 Discher et al. demonstrated vesicle formation from ethylene oxide/ethylethylene diblock copolymers and showed that these structures display mechanical stabilities 10 times greater than phospholipid-based structures.8 A few groups examined the self-assembly of ethylene oxide/ butylene oxide (EO/BO) diblock copolymers in aqueous solutions. However, although numerous phases were observed, no MLVs were reported.9-13 To the authors’ knowledge, the spontaneous formation of multilamellar vesicles from ethoxylated, nonionic diblock copolymers has not been reported. (7) Laibin, L.; Eisenberg, A. J. Am. Chem. Soc. 2001, 123, 1012. (8) Discher, B. M.; Won, Y. Y.; Ege, D. S.; Lee, J. C.-M.; Bates, F. S.; Discher, D. E.; Hammer, D. A. Science 1999, 284, 1143. (9) Pople, J. A.; Hamley, I. W.; Fairclough, J. P. A.; Ryan, A.; Komanschek, B. U.; Gleeson, A. J.; Yu, G.-E.; Booth, C. Macromolecules 1997, 30, 5721. (10) Kelarakis, A.; Havredaki, V.; Yu, G.-E.; Derici, L.; Booth, C. Macromolecules 1998, 31, 944. (11) Holmqvist, P.; Alexandridis, P.; Lindman, B. J. Phys. Chem. B 1998, 102, 1149. (12) Li, H.; Yu, G.-E.; Price, C.; Booth, C.; Hecht, E.; Hoffmann, H. Macromolecules 1997, 30, 1347. (13) Alexandridis, P.; Olsson, U.; Lindman, B. Langmuir 1997, 13, 23.
10.1021/la015714h CCC: $22.00 © 2002 American Chemical Society Published on Web 06/06/2002
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This article demonstrates the formation of MLVs from ethylene oxide/butylene oxide (EO/BO) diblock copolymers upon simple mixing with water. We selected these copolymers based on the premise that the BO monomer would produce a hydrophobic block with the volume-tolength ratio needed for vesicle formation. Examination of several EO/BO compositions with similar BO lengths showed a marked effect of EO block length on spontaneous MLV formation in water. We also explored how copolymer concentration, shear, sonication, thermal cycling, and the addition of electrolyte affect structure formation and size. Experimental Section Materials. Ethylene oxide (EO, 99.5+%), butylene oxide (BO, 99+%), toluene (HPLC grade), and potassium ethoxide (95%) were purchased from Sigma-Aldrich. Molecular sieves were added to the toluene upon receipt and to the BO approximately 24 h before use. Otherwise, the materials were used as received. Water was distilled and then filtered through a Barnstead (Newton, MA) NANOpure II water purification unit to achieve a minimum resistivity of 17.5 MΩ cm. Copolymer Synthesis. The EO/BO copolymers were synthesized and characterized at The Dow Chemical Company. The copolymers were prepared via sequential anionic polymerization of 1,2-butylene oxide and ethylene oxide. To do this, the potassium ethoxide initiator (12.63 g), dried toluene (250 g) and BO (110 g) were added to an oven-dried Parr Reactor (Parr Instruments, Moline, IL), which was then sealed. The reactor was purged with dry N2 for 5 min, blanketed with 30 psi of N2, and then heated to 110 °C with constant stirring. A controlled exothermic reaction reaching 120 °C was observed. The reactor was maintained at 120 °C for ∼4 h and then cooled to 5 °C (6 °C below the boiling point of EO), and liquid EO (75 g) was added. The reactor was reheated to 90 °C, and a second controlled exothermic reaction reaching 120 °C was observed and allowed to run to completion, ∼2 h. The reactor was cooled to room temperature and purged with N2, and half (240 g) of the product was removed. The reaction of the removed material was terminated by addition of 2 mL of hydrochloric acid (12 N), and the sample was set aside. A second aliquot of EO (18 g) was added, the reactor was blanketed with N2 and reheated to 90 °C. A third controlled exothermic reaction reaching 120 °C was observed and allowed to run to completion, ∼2 h. The reactor was cooled to room temperature and purged with N2, and the remaining product was removed. This reaction was also terminated with hydrochloric acid. The product was extracted using a toluene/3 M NaCl aqueous dispersion and isolating the organic phase. The solvent was removed by evaporation. The reaction produced 96 g of EO11BO11 and 82 g of EO19BO11 for a combined yield of greater than 85%. Other copolymer compositions were prepared in a similar manner with a slightly different workup procedure that included reaction quenching with acetic acid. Caution: EO is a suspected teratagen and should be handled with care. Copolymer Characterization. NMR spectroscopy (Varian Gemini 300) was performed to determine the copolymer composition using techniques previously described.14 The methyl protons on the initiator (λ) produced a unique peak at 1.24 ppm, while the methyl protons of the BO units (R) appeared at 0.92 ppm. The ratio of the two peaks (R/λ) showed the average number of BO units per chain. The EO block length was calculated from methylene signals (3.2-3.9 ppm) after subtracting the number of methylene protons on the initiator and BO block from the total number of methylene protons. Vesicle Preparation. One drop of the copolymer was added to a tared 5-dram vial, the vial was weighed, and the amount of water required for the desired concentration was added. The vial was moderately agitated by hand (∼1 shake s-1) for 30 s and allowed to stand for a minimum of 24 h before examination. Aqueous dispersions of each copolymer were generally prepared in concentrations ranging from 0.005 to 20 wt %, but the majority of work focused on 0.5, 1.0, 2.0, 5.0, and 10.0 wt % samples. This (14) Ribeiro, A. A.; Dennis, E. A. In Nonionic Surfactants; Physical Chemistry; Shick, M. J., Ed.; Marcel Dekker: New York, 1987; pp 975979.
Figure 2. Micrograph of spontaneously formed vesicles in a 5.0 wt % aqueous dispersion of EO11BO11. Micrograph taken using plane-polarized light at 100× magnification. concentration range was selected to ensure that the EO headgroups were fully solvated and to eliminate the effects of micellemicelle interactions.15 Samples were visually inspected for clarity, relative viscosity, and number and type of any mesophases present.16 Plane-Polarized Light Microscopy. Plane-polarized light microscopy (PLM) was used to determine type and extent of liquid crystal formation (Olympus Vanox transmission microscope, Olympus Optics, Tokyo). Six crystalline structures were identifiable, the most important being the spherical lamellae. (Numerous micrographs of these structures can be found in the literature, and the optical patterns are sufficiently distinct as to allow facile identification.16-23) Figure 2 is a PLM image of a typical sample containing vesicles and shows the Maltese cross patterns indicative of spherical lamellar structures. Light Scattering. Particle size distributions were obtained using a Coulter LS230 particle size analyzer (Beckman Coulter, Miami, FL) that can detect particles with diameters from 40 nm to 2000 µm. The instrument was fitted with the small volume module. Instrument performance was confirmed using 0.357 and 1.0 µm polystyrene latex standards, and all analyses were carried out using the “pslref” (polystyrene latex reference) model. Screening experiments showed that the dilution process used by this instrument did not alter the particle size distribution of the sample over the time involved. Particle sizes obtained in lightscattering experiments were consistent with MLV sizes viewed in PLM of undiluted samples. For particles smaller than 50 nm, a Brookhaven 90Plus (Brookhaven Instruments Corp., Holtsville, NY) particle size analyzer was used. Thermal Stability. Thermal transitions were monitored with PLM using a Linkam THS600 hot stage and a Linkam PR600 Controller (Linkam Scientific Instruments Ltd, U.K.). The sample holder consisted of a stainless steel washer glued to a glass slide with epoxy resin. After the dispersion was deposited into the center of the washer, a glass slide cover was glued to the washer to seal the holder. The samples were heated at 1 °C/min with a 10 min pause every 5 °C to allow for equilibration. (15) Mingvanish, W.; Mai, S.-M.; Heatley, F.; Booth, C.; Attwood, D. J. Phys. Chem. B 1999, 103, 11269. (16) Wanka, G.; Hoffmann, H.; Ulbricht, W. Macromolecules 1994, 27, 4145. (17) Yu, G.-E.; Li, H.; Fairclough, J. P. A.; Ryan, A. J.; McKeown, N.; Ali-Abid, Z.; Price, C.; Booth, C. Langmuir 1998, 14, 5782. (18) Kunieda, H.; Nakamura, K.; Davis, H. T.; Evans, D. F. Langmuir 1991, 7, 1915. (19) Gradzielski, M.; Muller, M.; Bergmeier, M.; Hoffman, H.; Hoinkis, H. J. Phys. Chem. B. 1999, 103, 1416. (20) Dorfler, H. D.; Knape, M. Tenside, Surfactants, Deterg. 1993, 30, 3. (21) Gray, G. W.; Windsor, P. A. Liquid Crystals and Plastic Crystals; Ellis Horwood: Sussex, England, 1974; p 223. (22) Franses, E. I.; Hart, T. J. J. Colloid Interface Sci. 1983, 94, 1, 1-13. (23) Collings, P. J. Liquid Crystals; Princeton University Press: Princeton, NJ, 1990; p 90.
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Table 1. Composition, Molecular Weight, Density, and Concentration Range for PLM-Detectable Vesicle Formation for EO/BO Diblock Copolymersa copolymer
Mn
density (g/mL)
EO6BO11 EO7BO12 EO11BO11 EO14BO10 EO19BO11 EO24BO10
1154 1222 1322 1384 1661 1824
1.00 1.01 1.01 1.02 1.03
concns for MLV formation (wt %) 0.05-20 0.05-20 0.05-20 0.5-20 2.0-20 none
a Values ranging from 0.05 to 20 wt % were examined for all copolymers. Some copolymers formed MLVs over a wider concentration range.
Cryogenic Scanning Electron Microscopy (Cryo-SEM). Cryo-SEM (JEOL JSM-6320FV) was used to ascertain whether the vesicles were unilamellar or multilamellar structures. The dispersions were flash frozen in liquid propane and stored in liquid nitrogen. They were then transferred to a cryo preparation chamber at -90 °C, fractured with a cooled scalpel, held at -90 °C for 2 min in a vacuum to sublime the surface ice, and sputter coated with conductive chromium. The samples were examined at -125 °C using a 3 keV electron beam.
Results Vesicle Formation. Effects of Composition. We examined vesicle formation by several EO/BO copolymers with similar BO block lengths, but different in EO chain length (Table 1). Copolymers with an EO length less than 15 spontaneously formed vesicles (as detected by PLM) at concentrations ranging from 0.05 to 20 wt % at room temperature. In contrast, EO19BO11 produced PLMdetectable vesicles only at concentrations above 2.0 wt % copolymer, and EO24BO10 showed no detectable vesicle formation in either PLM or light scattering experiments. Figure 2 shows a typical PLM image of EO11BO11 MLVs, and images for most other vesicle-forming compositions were similar. Copolymers with EO lengths less than 20 had cloud points 65 wt % of the copolymer was in these structures. In contrast, the yield of MLVs from 2.0 wt % EO19BO11 dispersions was less than 10%, again showing the greater propensity for vesicle formation by EO11BO11. Sedimentation of the particles is consistent with MLV formation because a multilayered structure should have a density slightly higher than water (Table 1). Unilamellar vesicles would be primarily composed of water and thus would be difficult to sediment. Given the densities in Table 1 and assuming that the copolymer occupies 50% of the volume of the sphere, an EO11BO11 MLV would have a density about 0.5% greater than water. This density difference, in addition to the large particle size, explains why MLV sedimentation occurs and makes centrifugation a plausible means of isolating large structures.24 Cryo-SEM. Because the Maltese cross patterns observed in PLM may result from either unilamellar or multilamellar vesicles, we examined dispersions using cryo-SEM to obtain more information about the structures. The micrograph in Figure 3 shows that the larger structures formed from EO11BO11 are multilamellar. The concentric layers are apparent from spots where the structure was punctured during freeze fracture. These structures were abundant and uniform in size. Particle Size Distribution and Stability. Because EO11BO11 readily formed MLVs over a wide range of concentrations, we conducted extended structural studies using this copolymer. Light scattering data from a 5.0 wt (24) Giddings, J. C. Unified Separation Science; Wiley-Interscience: New York, 1991; p 173.
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Figure 4. Particle size distribution of MLVs in a 5.0 wt % aqueous dispersion of EO11BO11 before and after various postformation treatments.
% EO11BO11 dispersion (Figure 4) showed that structure diameters ranged from 3.0 to 30 µm with a mean of 5.1 µm. Similar distributions occurred with 0.5, 1.0, and 2.0 wt % dispersions. Interestingly, we observed no particles with diameters between 0.04 and 2.0 µm. (On the basis of copolymer length, spherical micelles would be expected to have diameters less than 0.02 µm and would be undetectable in these measurements using the Coulter LS230.) Once formed, the particle size distribution (PSD) was relatively unaffected by moderate speed magnetic stirring, vortex mixing (Thermolyne vibrational mixer, model 37615), or mild sonication (Branson sonic bath, model 2200) (Figure 4). These techniques are routinely used to generate or alter the size of phospholipid MLVs, so we would not expect them to destroy the vesicle structures.4 However we were surprised that these treatments had so little effect on particle size. Dispersions of EO11BO11 were also thermally cycled to evaluate the effects of heating on PSD. Samples were heated to 85 °C for periods of 15-60 min, vigorously agitated by hand, and returned to the oven. This procedure was repeated until the samples had been heated for a total of 3 h. After thermal cycling, the samples were gently agitated and set aside to stand undisturbed for 24 h at room temperature. Light scattering data showed no significant difference between the PSD of an EO11BO11 dispersion before and after thermal cycling. In contrast, thermal cycling of EO19BO11 dispersions that did not spontaneously produce MLVs showed MLV formation (as determined by PLM) after heating. Dispersions of 0.5 and 1.0 wt % EO19BO11 that were thermally cycled showed vesicle formation with a PLM-determined size distribution similar to that for EO11BO11 samples (Figure 5). A 10.0 wt % EO19BO11 dispersion appeared unchanged by thermal cycling, showing the same PLM size distribution and apparent abundance of vesicles before and after heating. Thermal Stability of Liquid Crystalline Structures. Upon heating, the EO group undergoes dehydration and becomes increasingly hydrophobic.25,26 Thus, although EO/BO vesicle size distributions are stable to thermal cycling, MLVs may undergo phase or shape changes at high temperatures before cooling and returning to the (25) Bailey, F. E.; Koleske, J. V. In Nonionic Surfactants; Physical Chemistry; Shick, M. J., Ed.; Marcel Dekker: New York, 1987; pp 929935. (26) Clint, J. H. Surfactant Aggregation; Chapman and Hall: New York, 1992; pp 23 and 24.
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MLV structure. We examined structures from a 5.0 wt % dispersion of EO11BO11 as a function of temperature using PLM with a microscope hot stage. At room temperature liquid crystalline structures were evident by the presence of the Maltese cross pattern. These liquid crystalline structures persisted until the sample reached 38 °C. At that point the Maltese crosses disappeared, but nonpolarized microscopy showed that the structures maintained their spherical shape. No liquid crystalline structures were evident between 38 and 65 °C, the maximum temperature examined, and no coalescence or change in structure shape was observed. Upon cooling from 45 to 35 °C the Maltese cross patterns reappeared within 5 min. The same procedure was followed for structures formed from a 5.0 wt % EO19BO11 dispersion with similar results (phase transition ∼42 °C). Thus, there is no obvious redistribution of the block copolymers that make up the vesicles during this thermal cycling and the formation of liquid crystalline structures in the vesicles is thermally reversible. However, when dispersions are heated in an oven to temperatures above 75 °C, complete phase separation of the block copolymers from water is observed. Re-formation of liquid crystalline structures is achieved by gentle agitation of the two-phase system at room temperature. Extrusion. Extrusion is frequently used to reduce MLV sizes or convert MLVs to unilamellar vesicles.4 To evaluate the effect of extrusion, a 5.0 wt % dispersion of EO11BO11 was forced through a 5 µm Nylon filter repeatedly (7 times) using a syringe pump. Figure 6 shows the change in structure size due to extrusion and indicates that the mean particle diameter decreased from 5 µm to 70 nm. Greater than 95 wt % of the copolymer was recoverable after extrusion. The same initial dispersion (5.0 wt % EO11BO11) was also extruded through a 0.45 µm nylon filter with essentially the same results as with the 5.0 µm filter.27 This PSD after extrusion was stable for at least 2 months with no reversion to larger structures. Discussion Vesicle Formation Based on Surfactant Geometry. Israelachvili et al. described micellar structure formation in terms of a critical packing factor, Φ, defined in eq 1
Φ ) V/(lcao)
(1)
where V is the hydrophobe volume in the micelle core, ao is the cross sectional area of the hydrophilic headgroup at the micelle surface, and lc is the hydrophobic chain length.28This model hypothesizes that changes in the surfactant geometry, which are reflected in changes in V, lc, or ao, affect the type of micelle that the amphiphile may form. At a Φ value of 0.5, the model predicts formation of spherical lamellae. As the packing factor decreases (Φ approaches 0), the extent of curvature increases, and the structure shape moves toward spherical micelle. While this model is highly simplified, it provides important insights into the relationship between molecular geometry and micellar structures. We thought that preparation of the hydrophobic block from BO, which has a volume about 50% larger than a straight-chain aliphatic block of similar length, might increase hydrophobe volume sufficiently to form vesicles from EO/BO diblock copolymers. However, vesicle forma(27) As tortuosity may have some effect on extrusion, a 5.0 wt % dispersion of EO11BO11 was also extruded through a 0.4 µm tracketched polycarbonate membrane. This resulted in the same particle sizes as found with extrusion through the nylon membranes. (28) Israelachvili, J. N. Intermolecular and Surface Forces; Academic: New York, 1992; pp 380-384.
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Figure 5. The micrograph on the left shows a 1.0 wt % dispersion of EO19BO11 prepared at room temperature. The micrograph on the right shows the same dispersion after being thermally cycled at 85 °C for 3 h. (Details are included in the text.) Both micrographs were taken using plane polarized light at 100× magnification.
Figure 6. Particle size distribution before and after extruding a 5.0 wt % dispersion of EO11BO11 through a 5.0 or 0.45 µm filter.
tion is also dependent on the EO block because the headgroup size varies with EO length. Using the geometric model, we would expect that as ao increases (length of EO increases), the shape of the resulting structures would move from planar lamellae to spherical lamellae and finally to spherical micelles. In agreement with this trend we find that the copolymers with the shorter EO blocks (1 µm. MLVs spontaneously form in concentration ranges from 0.05 to 20 wt % for the copolymers with EO < 15, in concentrations of 2.0-20 wt % for the EO19BO11, and do not form for EO > 23. Initial particle size is independent of concentration for a given copolymer, and typical EO11BO11 structure yields are approximately 65% of the total amount of copolymer. The vesicular structures are mechanically stable and resistant to a variety of postformation treatments, but they can be extruded to form smaller structures. Additionally, both EO11BO11 and EO19BO11 vesicles are thermally stable to ∼40 °C, at which point they undergo a transition to a noncrystalline phase. Acknowledgment. The authors gratefully acknowledge the financial and technical support of The Dow Chemical Company and the assistance provided by Pat Green in synthesizing these materials. We thank Tom Kalantar, Stacie Erickson, Reed Shick, and Dave Williams for their assistance and ideas. Thanks also to Robert K. Prud’homme (Princeton University) for enlightening discussions on this topic. LA015714H
