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Polymerization of Equilibrium Vesicles John D. Morgan, Christopher A. Johnson, and Eric W. Kaler* Center for Molecular Engineering and Thermodynamics, Department of Chemical Engineering, University of Delaware, Newark, Delaware 19711 Received May 12, 1997. In Final Form: September 10, 1997X Spontaneous vesicles form in aqueous solutions of mixtures of cationic and anionic surfactants. Here we report that vesicles are also found in systems containing a polymerizable monomer as a fourth component and that these vesicles can be polymerized with retention of the equilibrium structure. This is a convenient and versatile approach in which the vesicle architecture can be controlled by equilibrium surfactant selfassembly prior to polymerization, while the microstructure of the spherical polymer network can be controlled by the choice of monomer and degree of cross-linking.
1. Introduction The formation of vesicles or liposomes is a familiar property of bilayer-forming surfactants, such as the phospholipids that constitute the major component of cell walls. These hollow spherical structures have found a variety of uses including encapsulation devices for the controlled release of drugs and microreactors for the synthesis of monodisperse nanometer-sized semiconductor particles and for the partitioning of reactants in artificial photosynthesis systems, inter alia.1-4 Typically vesicles are formed far from thermodynamic equilibrium through sonication or other mechanical disruption of bilayer phases, techniques which do not allow for close control of the vesicle size. These metastable vesicles ultimately revert to stable flat bilayer structures with resulting loss of integrity, limiting their use in many applications.5 One route to structural stabilization that has received much attention is polymerization of the vesicles, in order to lock into place the nonequilibrium structure. In most cases this has required the synthesis of specialty surfactants incorporating a polymerizable group such as a vinyl moiety, and the linear polymer that results is of low molecular weight with a correspondingly large number of chains per vesicle. An alternative approach is suggested by reports of the spontaneous formation of unilamellar vesicles in solutions of mixtures of cationic and anionic surfactants.6-8 The thermodynamics of the formation of these structures has received detailed attention,9-11 and unlike the metastable vesicles this class of vesicles is fully stable in solution. As demonstrated below, vesicles also form in these systems in the presence of a fourth componentsa hydrophobic polymerizable monomer. These monomer-laden vesicles are also equilibrium structures, and as such their dimensions are controlled by the thermodynamics of selfassembly. X Abstract published in Advance ACS Abstracts, November 1, 1997.
(1) Fendler, J. H.; Tundo, P. Acc. Chem. Res. 1987, 17, 3. (2) Fendler, J. H. Membrane-Mimetic Approach to Advanced Materials; Springer-Verlag: New York, 1994; Vol. 113. (3) Liposome Technology; Gregoriadis, G., Ed.; CRC Press: Boca Raton, FL, 1984. (4) Polymerization in Organised Media; Paleous, C. M., Ed.; Gordon and Breach Science Publishers: Philadelphia, PA, 1992. (5) Madani, H.; Kaler, E. W. Langmuir 1990, 6, 125. (6) Herrington, K. L.; Kaler, E. W.; Miller, D. D.; Zasadzinski, J. A.; Chiruvolu, S. J. Phys. Chem. 1993, 97, 13792. (7) Herrington, K. L., PhD thesis, University of Delaware, 1994. (8) Kaler, E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasadzinski, J. A. Science 1989, 245, 1371. (9) Brasher, L. L.; Kaler, E. W. Langmuir 1996, 12, 6270. (10) Yuet, P. K.; Blankschtein, D. Langmuir 1996, 12, 3802. (11) Yuet, P. K.; Blankschtein, D. Langmuir 1996, 12, 3819.
S0743-7463(97)00495-2 CCC: $14.00
In this article we demonstrate the construction of polymerized vesicles consisting of a single macromolecular shell (in the case of cross-linking monomers) within the bilayer of spontaneous vesicles. The structure of the resulting polymer network is amenable to control through the amount and identity of the monomer incorporated, and the degree of cross-linking, while the use of spontaneous vesicles offers the possibility of exploiting equilibrium self-assembly principles to control the vesicle size. This approach to the engineering of polymerized vesicles is extremely convenient in contrast to other popular techniques, requiring only the preparation of solutions of off-the-shelf surfactants and monomers with subsequent facile polymerization. We have characterized the vesicles and their polymerized counterparts formed in mixtures of the cationic surfactants cetyltrimethylammonium toluenesulfonate (CTAT) or dodecyltrimethylammonium bromide (DTAB) with the branched chain anionic surfactant sodium dodecylbenzenesulfonate (SDBS) using quasielastic light scattering (QLS), atomic force microscopy (AFM), and viscometry. Linear polystyrene has been recovered from the vesicles and the molecular weight distribution characterized using gel permeation chromatography with lowangle laser light scattering (GPC/LALLS). The polymerization of styrene in vesicles formed from dioctadecyldimethylammonium bromide using a similar technique has been reported;12,13 however, the monomer-containing vesicles in that case are of the metastable class and do not offer the advantages afforded by equilibrium vesicles. 2. Materials and Methods The surfactants DTAB and SDBS (TCI America) and CTAT (Sigma) and the cationic water-soluble initiator V-50 (2,2′-azobis(amidinopropane hydrochloride), Wako Chemical Co., USA) were used as received. The monomers styrene and divinylbenzene (DVB) were obtained from Aldrich and distilled prior to use to remove inhibitor. The DVB sample consisted of 50% DVB and 50% of isomers of ethylstyrene. Several techniques for preparation of mixed surfactant vesicle solutions have been described elsewhere.6 In this study monomerladen vesicle solutions were prepared by two methods. In the first method the monomer was dissolved in a micellar solution of the cationic surfactant, which was then mixed with a solution of the anionic surfactant under rapid stirring. The resulting bluish translucent solution was allowed a week for vesicles to equilibrate before further characterization. In the second method, solutions of the two surfactants were mixed with rapid stirring to form a vesicle solution which was equilibrated for a (12) Kurja, J.; Nolte, R. J.; Maxwell, I. A.; German, A. L. Polymer 1993, 34, 2045. (13) Murtagh, J.; Thomas, J. K. Faraday Discuss. Chem. Soc. 1986, 81, 127.
© 1997 American Chemical Society
6448 Langmuir, Vol. 13, No. 24, 1997 week. Aliquots (20 mL) of this solution were then added to a sample tube containing a small amount of the monomer and stirred at a high shear rate. The monomer takes several hours to dissolve, and stirring was continued for 24 h. A further 24 h was allowed before characterization. Vesicle radii were obtained by QLS using a spectrometer of standard design (Brookhaven Model BI-200SM goniometer and Model BI-9000AT correlator with a Lexel 300 mW 488 nm argon laser at a scattering angle of 90°) and data analyzed by the method of cumulants, assuming the vesicles behave as noninteracting hard spheres. Reported results are an average of five separate measurements of each sample. Large error bars are present in some measurements due to the presence of dust, as we wished to avoid perturbing the vesicles by filtration. Viscosity measurements were made using a capillary viscometer at 25.00 °C. For polymerizations the second preparation method was used to prepare 50 mL of a monomer-laden vesicle solution in a round bottom flask. This solution was degassed under vacuum to remove dissolved oxygen, and the polymerizations were performed at 60 °C for 24 h under a positive pressure of nitrogen. Polymerization was initiated by the addition of 0.01 g of solid V-50 through a small port. Polymer was precipitated from solution by addition of acetone and redissolved in toluene, and residual surfactant was extracted by washing with water. The toluene solution was dried down and the polymer dissolved in methyl ethyl ketone at 1.0 mg/mL for GPC/LALLS analysis. Calibration constants for the LALLS and refractive index detector were obtained using polystyrene standards (Polymer Laboratories). AFM imaging of the vesicles was performed in liquid using a Nanoscope III Multimode scanning probe microscope (Digital Instruments, Santa Barbara, CA).14 A solution of the liquid sample was introduced into a glass fluid cell containing the AFM probe and a freshly cleaved mica substrate (Electron Microscopy Sciences, Fort Washington, PA) mounted on the piezoelectric scanner. After allowing several minutes for the vesicles to adsorb to the mica substrate the cell was flushed with distilled water and the sample imaged by liquid tapping mode AFM using an oxide-sharpened silicon nitride probe (oriented twin tip probe, Digital Instruments). Details of the technique are given elsewhere.14,15
3. Characterization of Unpolymerized Vesicles It is necessary to show firstly that the monomer resides in the bilayer shell, rather than in other micellar structures that might conceivably be formed by surfactant rearrangement, and secondly that the polymer is formed within the bilayer with retention of the equilibrium structure. The second issue is particularly important as polymerization in microstructured fluids, such as microemulsions, typically leads to the loss of the initial equilibrium structure as surfactant and monomer are redistributed during the course of the polymerization reaction.16-18 The phase diagrams of the CTAT/SDBS/H2O and DTAB/ SDBS/H2O systems have been reported elsewhere.6-8,19 Spontaneous vesicles form in these solutions below about 5 wt % surfactant and may be positively or negatively charged depending on the ratio of surfactants. The systems chosen for this study contained a total of 1 wt % surfactant in various mixing ratios. Monomer-laden vesicles were prepared either by mixing two solutions of the individual surfactants, one of which contained micellized monomer, or by addition of monomer to previously prepared vesicle solutions. In each case translucent, (14) Hansma, P. K.; et al. Appl. Phys. Lett. 1994, 64, 1738. (15) Johnson, C. A.; Lenhoff, A. M. J. Colloid Interface Sci. 1996, 179, 587. (16) Holtzscherer, C.; Wittmann, J. C.; Guillon, D.; Candau, F. Polymer 1990, 31, 1978. (17) Pe´rez-Luna, V. H.; Puig, J. E.; Castan˜o, V. M.; Rodriguez, B. E.; Murthy, A. K.; Kaler, E. W. Langmuir 1990, 6, 1040. (18) Thundathil, R.; Stoffer, J. O.; Friberg, S. E. J. Polym. Sci., Polym. Chem. Ed. 1980, 18, 2629. (19) Kaler, E. W.; Herrington, K. L.; Murthy, A. K.; Zasadzinski, J. A. J. Phys. Chem. 1992, 96, 6698.
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Figure 1. Vesicle radius vs wt % oil for 1 wt % surfactant in H2O: ([) CTAT:SDBS in 7:3 weight ratio (middle of vesicle phase), oil is DVB; (O) CTAT:SDBS in 1:1.07 weight ratio (near phase boundary), oil is DVB; (0) CTAT:SDBS in 1:1.07 weight ratio, oil is styrene.
bluish solutions resulted with a similar QLS radius. That vesicles resulted in each case suggests that swollen vesicles are indeed the free energy minimizing structure. Only the second method of preparation was used in subsequent work. The vesicles swell upon addition of monomer and are capable of taking up varying amounts of oil before phase separation occurs, with samples in the middle of the positively-charged vesicle region of the phase diagram being able to accommodate more oil than those near the phase boundary. Figure 1 shows the size of CTAT/SDBS vesicles as a function of the amount of added monomer. Vesicles prepared at a weight ratio of 1.07/1 CTAT/SDBS at 1.0 wt % total surfactant concentration are close to the vesicle/lamellar phase boundary. These vesicle solutions only absorb roughly 6 wt % of oil with respect to surfactant before the apparent size increases catastrophically. The larger vesicles are unstable and the solutions ultimately flocculate. Vesicles containing surfactant in the weight ratio 7/3 CTAT/SDBS at 1.0 wt % total concentration are in the middle of the positively-charged vesicle lobe of the phase diagram. Addition of up to 30 wt % oil with respect to surfactant to these vesicles leads to only a slight increase in the apparent vesicle size, and the resulting structures have been stored for several months without destabilization. Addition of larger amounts of oil leads to the formation of flocs. Figure 2 shows more detail of the size variation of the 7/3 CTAT/SDBS vesicles as monomer is added. The growth on addition of oil could be due either to the rearrangement of surfactant to a new equilibrium architecture consisting of a smaller number of larger vesicles or to swelling of the existing vesicles without significant surfactant redistribution. The increase in size was calculated in the latter case assuming that the vesicles swell slightly to accommodate the added volume of monomer within the hydrophobic region of the bilayer. Consider the empty vesicle as a spherical bilayer of thickness b + 2h, where b is the thickness of the hydrocarbon chain region and h is the thickness of the headgroup region. The mass mv of a single unswollen vesicle is
mv )
4πMw(Ro2 + (Ro - b - 2h)2) ahNA
(1)
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Figure 2. Detail of Figure 1, now showing radius vs wt % oil with respect to surfactant. The error bars are obtained from repeated measurements of a single sample. The solid line is determined using the isotropic dilation model (eq 7) with ah ) 48 Å2, b ) 25 Å, and h ) 3 Å.
where ah is an average area per headgroup for the surfactant mixture, Ro is the radius of the outer layer, which is reported by QLS, NA is Avogadro’s number, and Mw is the average surfactant molecular weight, given by
Mw ) R1m1 + R2m2
(2)
where Ri and mi are the mole fractions and molecular weights, respectively, of each surfactant. The volume Vo of the hydrocarbon region is in the unswollen vesicle is
Vo )
4π [(Ro - h)3 - (Ro - b - h)3] 3
(3)
Let f be the weight fraction of added monomer with respect to surfactant. The change in volume of the vesicle bilayer is then
dV ) fmv/F
(4)
where F is the monomer density. Assume that monomer is excluded from the headgroup regions and that the hydrocarbon region swells uniformly to accommodate this added volume; i.e., lengths in the hydrocarbon region are scaled by a factor 1 + s. This scaling gives
(1 + s)3 ) 1 +
dV Vo
(5)
and
R(f) - h ) (1 + s)(Ro - h) Consequently
(
R(f) - h ) (Ro - h) 1 +
(6)
)
3f(RcMc + RaMa) Ro2 + (Ro - b - 2h)2 FahNA (R - h)3 - (R - b - h)3 o
o
1/3
(7)
gives R(f), the radius of the swollen vesicle as a function of added monomer. ah has been measured for CTAT/SDBS mixtures by surface tensiometry, yielding a value of 48 Å2, independent
Figure 3. Dilution of polymerized (0) and unpolymerized (b) vesicle samples. Solutions contain 0.725/0.275/0.1/99 DTAB/ SDBS/DVB/H2O by weight. A dilution factor of 1.0 corresponds to no dilution.
of the mixing ratio.7 The thickness of the hydrocarbon region has been measured by small angle neutron scattering for CTAB/sodium octyl sulfate vesicles at a mixing ratio of 3/7 by weight, respectively, giving a value of 22 Å.9 In our system we have a larger amount of the longchain surfactant, and the shorter surfactant contains a branched dodecylbenzenesulfonate chain, so the thickness should be somewhat greater. We assume a value for b of 25 Å and adopt a value for h of 3 Å. Using these values in eq 7, along with the measured value for Ro of 61.7 nm, this model is in good agreement with the experimental swelling data (Figure 2). This suggests that the monomer is taken up by the vesicle bilayer without significant redistribution of surfactant among the vesicles. 4. Characterization of Polymerized Vesicles Polymerizations were performed on samples containing 1 wt % surfactant and 10 wt % monomer with respect to surfactant. The visual appearance of the solutions and the radius of the vesicles measured by QLS were unchanged upon polymerization. Polymerizations have been performed with positively and negatively charged vesicles, and the resulting structures are stable, with no indication of phase separation or coagulation over several months. The polymerized structures are resistant to lysing with the detergent Triton X-100, unlike the unpolymerized systems which revert to a transparent micellar solution after addition of detergent. The radii of the polymerized vesicles also remain unchanged on addition of Triton X-100 (Figure 3). Dilution of the unpolymerized systems results in an increase in size (Figure 3), which we presume to be due to a change in the vesicle bilayer composition resulting from the selective removal of the more soluble surfactant.9-11 This is a feature of equilibrium vesicles not displayed by similar nonequilibrium vesicles and may be used to confirm that polymerization has indeed occurred in the bilayer. The polymerized vesicles show no change in size upon dilution as the polymer network is not capable of relaxation to the larger size. At high dilutions the polymerized vesicles become unstable due to loss of surfactant. A measure of the total volume of the vesicles in solution, free of artifacts due to polydispersity, is available through measurement of the solution viscosity as a function of dilution. The viscosity of a dilute solution of noninter-
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Figure 4. Intrinsic viscosities of diluted polymerized vesicle samples. The initial sample was the same as that of Figure 3. The line is a linear regression. A dilution factor of 1.0 corresponds to no dilution.
acting hard spheres is given by the Einstein equation20
η/η0 ) 1 + 2.5φ
(8)
where η is the suspension viscosity, η0 is the solvent viscosity, and φ is the volume fraction of spheres. The viscosities of the polymerized DTAB/SDBS/DVB vesicle solutions were determined at various dilutions with distilled water using capillary viscometry (Figure 4). The viscosity was linear in concentration, validating the noninteracting hard sphere model used in the interpretation of the QLS results. Fitting the data to eq 8 yielded a volume fraction of φ ) 8.7% for the undiluted sample. φ can also be calculated from the QLS radius for this sample of 81 nm, with the bilayer and surfactant parameters used previously in eq 1. A value of 9.1% results in good agreement with the viscometry data considering that polydispersity is not taken into account in calculation of the QLS radius. The polymerized vesicles could be directly imaged under water using atomic force microscopy in liquid tapping mode (Figure 5). The polymerized and cross-linked sample contained DTAB/SDBS/DVB/H2O at a composition of 0.725/0.275/0.1/99 by weight. The images show a polydisperse distribution of hemispherical structures, with the larger particles having a characteristic size in agreement with the QLS result. Attempts to image the unpolymerized vesicles in water under the same conditions were unsuccessful due to the fragility of these structures. AFM therefore provides a tool for discriminating between polymerized and unpolymerized vesicles. We also obtained freeze fracture electron micrographs of the polymerized vesicle solutions (not shown) which are similar to those already published for the neat surfactant system.8 Vesicles are clearly present, but it is not possible to discriminate between polymerized and unpolymerized structures on the basis of such images. Analysis of the AFM data provides several pieces of evidence for the hollowness of these structures. First, the structures are much larger than the solid latex particles formed in microemulsion polymerizations,17 so it is unlikely that the polymer has grown as a compact ball. Additionally, the adsorbed structures appear in the AFM image to be hemispherical, even when the tip(20) Hunter, R. J. Foundations of Colloid Science; Oxford University Press: Oxford, 1986; Vol. 1.
Figure 5. AFM image of polymerized vesicles under distilled water, deposited on mica surface. The image size is 1.0 µm × 1.0 µm, and the composition is the same as for Figure 3.
Figure 6. Molecular weight distribution W(M) vs M from GPC of polystyrene recovered from polymerized vesicle solution, composition 0.725/0.275/0.1 wt % DTAB/SDBS/styrene.
broadening artifact of the imaging process is considered.21 A height of 42 nm and a diameter of 143 nm measured for one of these objects is typical. These dimensions exclude the possibility of the polymerization resulting in solid particles, as the polymer in this sample is highly crosslinked and below the glass transition temperature. A solid spherical particle formed from this polymer would be quite rigid and present a height equal to its diameter, while a hollow spherical membrane of cross-linked poly-DVB should retain sufficient flexibility to deform upon adsorption and present the observed profile. Some coalescence is evident as a consequence of dilution within the AFM fluid cell, and alignment of the aggregates is due to the flow field present as distilled water is flushed through the cell. A number of very small particles are also evident; these may be either very small vesicles or solid latex particles formed through nucleation in the aqueous phase. (21) Yang, J.; Mou, J.; Shao, Z. Biochim. Biophys. Acta 1994, 105, 1199.
Polymerization of Equilibrium Vesicles
The molecular weight distribution of linear (un-crosslinked) polystyrene formed in the system DTAB/SDBS/ styrene/H2O (composition 0.725/0.275/0.1/99 by weight) was analyzed using GPC with IR and laser light scattering detection. The weight distribution W(M) of the polymer so obtained is remarkably monodisperse (Figure 6). Most of the polymer appears as a sharp peak at a degree of polymerization of about 2600 monomer units per chain, corresponding to roughly six linear chains per vesicle. A small peak also occurs at a higher degree of polymerization which contains about 2% of the total polymer. This peak may correspond to polymer formed in small particles through homogeneous nucleation or phase separation from the vesicle bilayer. In contrast, the greatest degree of polymerization reported for vesicles formed from polymerizable surfactants is approximately 500 monomer units.22 In our vesicles prepared with DVB the polymerized structure is certainly composed of a single crosslinked polymer molecule.
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These results support the contention that equilibrium vesicles may be polymerized with retention of their equilibrium structure. Vesicle polymerization by this technique is convenient and versatile and offers the potential of controlling all aspects of the polymerized vesicle architecture and properties through appropriate choice of surfactants, monomers, co-monomers, and polymerization conditions. This degree of structural control is expected to be particularly valuable in using vesicles as nanoencapsulation devices in a variety of applications. Acknowledgment. The authors thank Mike Kennedy of the University of California, Santa Barbara, for obtaining freeze-fracture electron micrographs. This work was supported by the National Science Foundation (CTS-9319447). LA970495E (22) Dorn, K.; Patton, E. V.; Klingbiel, R. T.; O’Brien, D. F.; Ringsdorf, H. Macromol. Chem., Rapid Commun. 1983, 4, 513.
