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Highly Stable Polymerizable Vesicles in Anionic Surfactant/ Ammonium Salt Mixtures in the Presence of Cross-linking Monomers for Convenient Preparation of Hollow Nanospheres Kenichi Oyaizu,† Yuichi Shiba,‡,§ Yukiaki Nakamura,‡ and Makoto Yuasa*,†,‡ Institute of Colloid and Interface Science, Tokyo UniVersity of Science, Tokyo 162-8601, Japan, Department of Pure & Applied Chemistry, Faculty of Science and Technology, Tokyo UniVersity of Science, Noda 278-8510, Japan, and Corporate Research Center, Mitsubishi Paper Mills Limited, Tokyo 125-8525, Japan ReceiVed December 13, 2005. In Final Form: February 13, 2006 Spontaneous and stable vesicles are formed from vinylbenzyltrimethylammonium chloride (VBTAC) and sodium dodecyl sulfate (SDS) ranging in molar composition from 3:7 to 7:3 in the presence of 30 mol % divinylbenzene (DVB). Dynamic light scattering analysis and transmission electron microscope observations revealed that microparticles with diameters around 120 nm (30%) and 580 nm (70%) were formed. Trapping efficiency of the vesicles examined with D-(+)-glucose amounted to ca. 15%. These vesicles were capable of undergoing polymerization in the presence of water-soluble radical initiators such as 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride to fix the vesicular structure. Retention of the vesicle size and bimodal size distribution characteristics during the polymerization indicated that intravesicular polymerization prevailed over the intervesicular process. SDS was stably retained in the cross-linked vesicle and was not removed from the vesicle by exhaustive dialysis or ultrafiltration, due to the electrostatic interaction within the cross-linked polymeric framework produced from VBTAC and DVB. The resulting hollow nanospheres are readily redispersed in water without the aid of additional surfactants.
Polymerization reactions in spherically closed amphiphilic bilayers,1-3 i.e., vesicles and liposomes,4-9 are known to fix the vesicular structure to provide stabilized hollow colloidal nanoparticles.10-13 A number of unsaturated phospholipids have been synthesized for stabilization of liposomes.14,15 Polymerization of metastable vesicles, typically formed by the dispersion of bilayer phases of double-chained surfactants incorporating * Corresponding author. M. Yuasa: Department of Pure & Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, Noda 278-8510, Japan. Fax: +81-4-7121-2432. Phone: +81-4-7124-1501. E-mail:
[email protected]. † Institute of Colloid and Interface Science, Tokyo University of Science. ‡ Department of Pure & Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science. § Corporate Research Center, Mitsubishi Paper Mills Limited. (1) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1988, 27, 113-158. (2) Lamparski, H. G.; Oblinger, E.; O’Brien, D. F. Macromolecules 1999, 32, 5450-5452. (3) Sisson, T. M.; Srisiri, W.; O’Brien, D. F. J. Am. Chem. Soc. 1998, 120, 2322-2329. (4) Yuasa, M.; Oyaizu, K.; Horiuchi, A.; Ogata, A.; Hatsugai, T.; Yamaguchi, A.; Kawakami, H. Mol. Pharm. 2004, 1, 387-389. (5) Nishide, H.; Yuasa, M.; Hashimoto, Y.; Tsuchida, E. Macromolecules 1987, 20, 459-461. (6) Tsuchida, E.; Nishide, H.; Yuasa, M.; Hasegawa, E.; Eshima, K.; Matsushita, Y. Macromolecules 1989, 22, 2103-2107. (7) Tsuchida, E.; Nishide, H.; Yuasa, M.; Babe, T.; Fukuzumi, M. Macromolecules 1989, 22, 66-72. (8) Yuasa, M.; Nishide, H.; Tsuchida, E.; Yamagishi, A. J. Phys. Chem. 1988, 92, 2987-2990. (9) Imura, T.; Gotoh, T.; Otake, K.; Yoda, S.; Takebayashi, Y.; Yokoyama, S.; Takebayashi, H.; Sakai, H.; Yuasa, M.; Abe, M. Langmuir 2003, 19, 20212025. (10) Lestage, D. J.; Urban, M. W. Langmuir 2005, 21, 4266-4267. (11) Kazakov, S.; Kaholek, M.; Kudasheva, D.; Teraoka, I.; Cowman, M. K.; Levon, K. Langmuir 2003, 19, 8086-8093. (12) Kazakov, S.; Kaholek, M.; Teraoka, I.; Levon, K. Macromolecules 2002, 35, 1911-1920. (13) Wu, J.; Lizarzaburu, M. E.; Kurth, M. J.; Liu, L.; Wege, H.; Zern, M. A.; Nantz, M. H. Bioconj. Chem. 2001, 12, 251-257. (14) Cheng, Q.; Stevens, R. C. Langmuir 1998, 14, 1974-1976. (15) Ohno, H.; Ogata, Y.; Tsuchida, E. Macromolecules 1987, 20, 929-933.
polymerizable groups, lock even the nonequilibrium vesicular structures into place, preventing them from reverting to more stable flat bilayers.16 A number of amphiphilic cross-linking monomers have been explored to circumvent the problem of intravesicular phase separation that could induce significant deformation of vesicles upon polymerization.17-19 Divinylbenzene (DVB) is known to swell the vesicle bilayers of various double-chained surfactants to act as a simple cross-linking agent.16,20 Polymerization of unilamellar vesicles, spontaneously formed by the mixing of single-chained cationic and anionic surfactants incorporating polymerizable groups, has also been reported.21 Incorporation of DVB into the hydrophobic bilayer of the spontaneous vesicles provide hollow spheres after polymerization. On the other hand, one could anticipate that a combination of vinylbenzyltrimethylammonium chloride (VBTAC) and DVB would allow spontaneous formation of polymerizable vesicles with a wide variety of anionic surfactants such as sodium dodecyl sulfate (SDS). Although various spontaneous vesicles obtained from polymerizable anion/cation surfactants have been reported, those containing a significant amount of SDS are unprecedented. Such systems have not been explored extensively, due probably to the difficulties in finding suitable compositions to form stable vesicles. Here, we report that spontaneous and stable vesicles are formed at certain composition ranges and that these vesicles are capable of undergoing polymerization to fix the vesicular structure (Scheme 1). The resulting hollow nanoparticles have resistance to organic (16) Jung, M.; den Ouden, I.; Montoya-Gon˜i, A.; Hubert, D. H. W.; Frederik, P. M.; van Herk, A. M.; German, A. L. Langmuir 2000, 16, 4185-4195. (17) Im, J.-Y.; Kim, D.-B.; Lee, S. H.; Lee, Y.-S. Langmuir 2003, 19, 63926396. (18) Liu, S.; O’Brien, D. F. Macromolecules 1999, 32, 5519-5524. (19) Sisson, T. M.; Lamparski, H. G.; Ko¨lchens, S.; Elayadi, A.; O’Brien, D. F. Macromolecules 1996, 29, 8321-8329. (20) Yang, W. Y.; Lee, Y.-S. Langmuir 2002, 18, 6071-6074. (21) Liu, S.; Gonza´lez, Y. I.; Kaler, E. W. Langmuir 2003, 19, 10732-10738.
10.1021/la053369g CCC: $33.50 © 2006 American Chemical Society Published on Web 05/05/2006
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Scheme 1. Concept of Polymerization in Spontaneous Vesicles Prepared from Aqueous VBTAC/SDS Mixtures in the Presence of a Cross-linking Agent DVB as Templating Strategies for Polymeric Hollow Spheres
Figure 1. (a) Aqueous dispersion of a mixture of 60 mM VBTAC, 40 mM SDS, and 30 mM DVB at room temperature before (a) and after (b) the polymerization (see Experimental Section for details), and the freeze-dried sample of the polymerized vesicle (c).
solvents such as acetone, hexane, chloroform, benzene, and toluene due to the cross-linked structure, and yet they are easily redispersed in water without the aid of additional surfactants. Experimental Section 2,2′-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA044), 2,2′-azobis{2-[1-(2-hydroxyethyl)-2-imidazolin-2-yl]propane} dihydrochloride (VA-060), 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) (V-70), 2,2′-azobis(isobutyronitrile) (AIBN), cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide (DTAB), stearyltrimethylammonium bromide (STAB), dioctadecyldimethylammonium chloride (DODAC), sodium hexadecyl sulfate (SHS), sodium oleate (SOA), and SDS were obtained from Wako Chem. and used as-received. DVB containing 20% 3and 4-ethylvinylbenzene, d-(+)-glucose, and VBTAC were obtained from Aldrich. All solvents were purified by distillation in the usual manner prior to use. Spontaneous vesicles were prepared from VBTAC, SDS, and DVB by simply mixing them in water. In a typical procedure, a mixture of VBTAC (0.127 g, 6.0 × 10-4 mol) and SDS (0.115 g, 4.0 × 10-4 mol) in water (10 mL) was stirred using a Voltex mixer for 10 s. To the mixture was added DVB (0.039 g, 3.0 × 10-4 mol), which was then stirred for an additional 10 s to produce a pale blue dispersion liquid. After the dispersion liquid was diluted 10-fold with water, polymerization was carried out by adding VA-044 (0.0015 g, 4.5 × 10-5 mol) under argon and stirring the mixture at 50 °C for 1 h. A fraction of SDS that was not taken up into the polymerized vesicle was removed by ultrafiltration on a polyethersulfone membrane (Millipore) with a pore size of 25 nm, and the resulting particles were purified by dialysis against H2O using seamless cellulose tubing (UC24-32-100) from Sanko Chem. Lyophilization of the dispersion liquid afforded the product as a white powder. Yield: 75 wt %. IR (KBr, cm-1): 1219 (νSO3). CP/MAS (ppm): δ 13.8, 22.7, 29.7, 31.6, 51.8, 126.5, 133.1. Trapping efficiency of the unpolymerized vesicles was evaluated by preparing the vesicle in an aqueous solution of D-(+)-glucose (0.2 M), followed by dialysis against aqueous NaCl (0.1 M) at 0 °C for 12 h using the cellulose tubing to remove the unencapsulated glucose. The amount of glucose entrapped in the vesicle was analyzed by the conventional mutarotase GOD method22 after disrupting the vesicle with ethanol. In transmission electron microscope (TEM) observations, the dispersion liquid containing the unpolymerized vesicles was quickly frozen in liquid propane. The frozen sample was fractured using a freeze-replica-making apparatus (Hitachi FR-7000A) at -150 °C. The fractured surface was replicated by evaporating platinum at an angle of 45°, followed by carbon at normal incidence to strengthen the replica. After the replica was washed with acetone and water, it was placed on a 150 mesh copper grid and observed with a TEM (22) Otake, K.; Imura, T.; Sakai, H.; Abe, M. Langmuir 2001, 17, 3898-3901.
(JEOL JEM-1200EX). The TEM image of the polymerized vesicle was obtained similarly. CP/MAS spectra were obtained using a JEOL JMN-ECP-300 spectrometer. 13C NMR spectra were recorded on a JEOL JNMLA300 spectrometer with chemical shifts downfield from tetramethylsilane as the internal standard. UV-vis spectra were recorded on a Shimadzu UV-2100 spectrometer. Dynamic light scattering (DLS) measurements were carried out using a NICOMP 380ZLS Particle Sizing System. Sample solutions were filtered prior to light scattering experiments using a syringe filter (PTFE filter media) with a pore size of 0.45 µm from Whatman Inc. The intensity of the scattered light at 90° was recorded in a homodyne experiment as a function of time and processed to obtain the autocorrelation function of the homodyne signal. A constrained linear least-squares fit procedure applied to the autocorrelation function gave relative errors of the fit parameters of 2.608-4.056% with zero residuals. The sufficiently small fitting errors of less than 5% gave a reliable distribution of translational self-diffusion coefficients. The hydrodynamic diameters (Dh) of the particles were obtained from the diffusion coefficients according to the Stokes-Einstein equation. IR spectra were obtained using a JASCO FT/IR-410 spectrometer and a KBr pellet. Lyophilization was carried out using a Kyowa Triomaster II A-04 freeze-drying system. Scanning electron microscopy (SEM) observations were carried out using a JEOL JSM6700F NT FE-SEM system.
Results and Discussion The use of spontaneous vesicles from mixtures of singlechained cationic and anionic surfactants, without the need to sonicate or mechanically disrupt bilayer phases, offers the possibility of using equilibrium self-assembly principles to control the vesicle size. To develop the spontaneous vesicles that undergo radical polymerization upon treatment with external initiators, aqueous VBTAC/SDS mixtures with a total concentration of 100 mM were explored, which ranged in molar composition from 0:10 to 10:0 in the presence of 30 mM DVB as a crosslinking agent. Almost transparent colloidal dispersions were obtained, however, from mixtures ranging in compositions from 0:10 to 2:8, suggesting that DVB was incorporated into the micelles of SDS when the amount of VBTAC was insufficient to form vesicles. On the other hand, DVB was separated from the aqueous phase in mixtures with excess VBTAC ranging in compositions from 8:2 to 10:0. Homogeneous pale blue dispersion liquid, as shown in Figure 1a, was successfully obtained for specific compositions ranging from 3:7 to 7:3. The dispersion with a composition of VBTAC:SDS ) 6:4 was especially stable, which remained intact at room temperature for several months and did not undergo further phase separation at temperatures ranging from 5 to 90 °C. Figure 2 shows the TEM picture of the resulting nanoparticles, which are spherical with diameters around 200 nm and are highly
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Figure 2. TEM picture of vesicles prepared by mixing 60 mM VBTAC, 40 mM SDS, and 30 mM DVB in water at room temperature, and a corresponding DLS histogram showing bimodal distribution of the vesicle size with average hydrodynamic diameters (Dh) near 120 and 580 nm.
dispersed in water. The TEM image suggests that the major particles are most likely unilamellar vesicles. In Figure 2 is also shown the dynamic light-scattering (DLS) histogram obtained for the same dispersion liquid. The particle is featured by a bimodal distribution of Dh centered at 120 nm (30%) and 580 nm (70%), which is consistent with the results of the TEM observations. The statistical accuracy of the bimodal character of the size distribution was confirmed by several independent measurements on different samples from the same stock solution. It is likely that the vesicles are surrounded by a small amount of undersized mixed micelles. The presence of the mixed micelles in the equilibrated mixture is equivocal by TEM observations and DLS measurements, but suggested by the liberation of a fraction of SDS during the ultrafiltration of the mixture after polymerization of the vesicle (see Experimental Section). DLS measurements revealed that the vesicles survived without alternation of the size distribution up to 10-fold dilution with water, but that they readily collapsed to form a homogeneous solution upon addition of organic solvents such as ethanol. Added support for the vesicle formation in water was provided by a substantial trapping efficiency examined with D-(+)-glucose, which amounted to 15%. In control experiments, mixtures of VBTAC and SDS without DVB gave only micellar solutions for all compositions, with trapping efficiencies of only 3.5% and a mean Dh of 27 nm. Even smaller trapping efficiencies of 1.2% and a mean Dh of 7.3 nm were found for a micelle by SDS. These results suggested that DVB contributed to the formation of vesicles by swelling the hydrophobic part of the bilayers to reduce their curvature. Having optimized the composition to give highly stable and thus apparently equilibrated vesicle, we examined the radical polymerization of the vesicle to fix the vesicular structure. The vesicle composed of VBTAC and SDS embedded with DVB underwent efficient radical polymerization in the presence of 5 mol % water-soluble initiators such as VA-044 and VA-060 under argon. The progress of the polymerization was monitored using the absorption band near 250 nm in the UV spectra of the dispersion liquid, due to the π-π* transition of the arylvinyl groups in VBTAC and DVB. The band decreased down to a few percent in intensity within 30 min, and almost disappeared after completion of the polymerization. The hydrophobic vinyl group in VBTAC is considered to be situated in proximity to those in DVB incorporated in the hydrophobic part of the vesicle bilayer, as illustrated in Scheme 1. Consumption of both monomers suggests that these monomers are uniformly distributed in the vesicle bilayer and that no phase separation occurs during the random copolymerization to produce a cross-linked polymer. The conventional water-insoluble initiators such as AIBN and V-70, which were probably incorporated into the hydrophobic layer of the vesicles, worked rather inefficiently, resulting in the
Figure 3. CP/MAS spectra for the freeze-dried sample of an aqueous VBTAC/SDS mixture with a molar composition of 6:4 (blue line) and the isolated nanoparticle in Figure 1c (red line).
presence of a substantial amount of unreacted vinyl monomers remaining in the dispersion liquid even after prolonged time of polymerization. Intervesicular polymerization was successfully suppressed by the 10-fold dilution of the pristine dispersion liquid in Figure 1a prior to the polymerization (vide infra), resulting in a white dispersion of the polymerized vesicle as shown in Figure 1b. Purification of the product was accomplished by exhaustive ultrafiltration and dialysis to remove the unreacted low molecular-weight components from the resulting liquid. In contrast to the pristine vesicle before the polymerization, the polymerized particle did not dissolve but readily dispersed in organic solvents such as acetone, hexane, chloroform, benzene, and toluene due to the cross-linked structure. After freeze-drying, the product was obtained as a white powder in 75 wt % yield as shown in Figure 1c. In control experiments, polymeric particles obtained from the VBTAC/SDS mixture containing styrene in place of DVB readily dissolved in the organic solvents, which demonstrated the efficacy of cross-linking within the polymerized vesicle for the solvent resistance. On the other hand, products from the polymerization of micelles obtained from VBTAC and SDS without the additional vinyl monomers were soluble even in water. Figure 3 shows the CP/MAS spectrum of the isolated polymerized vesicle, together with that of a freeze-dried powdery sample of an aqueous VBTAC/SDS mixture without volatile DVB. The peaks at 113 and 136 ppm, observed for the freezedried dispersion, were ascribed to the resonances from vinyl carbons in VBTAC, based on the coincidence with the 13C NMR chemical shifts of the corresponding peaks for VBTAC in CD3OD which appeared near 117 and 137 ppm, respectively. The vinyl carbons decreased in amount after the polymerization in the presence of DVB, which was confirmed by the absence of these peaks in the CP/MAS spectrum of the polymerized vesicles (Figure 3). Resonances from the vinyl carbons in DVB were not found in the spectrum, which demonstrated that DVB acted as an efficient cross-linking agent in the polymerized vesicle. Figure 4 shows the IR spectral changes upon the polymerization. A high-yielding conversion of the vinyl monomers to the crosslinked polymer was also suggested by the significant decrease in the intensity of the band at 3020 cm-1 due to the alkenyl C-H stretching vibration. The CP/MAS spectrum (Figure 3) also revealed the presence of SDS in the polymerized vesicle, which showed resonances
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Figure 5. SEM picture of the polymerized vesicles before the purification process, and corresponding DLS histogram showing the bimodal size distribution with average Dh near 290 and 1360 nm.
Figure 4. IR spectra for the freeze-dried mixture of VBTAC and SDS with a molar composition of 6:4 (a), and the isolated polymerized vesicle in Figure 1c (b).
near 30 ppm due to the alkyl carbons. In the IR spectra (Figure 4), an intense band at 1219 cm-1 was ascribed to the stretching of hydrated SO3- group (i.e., hydronium sulfate), which typically appeared near 1120-1230 cm-1. Interestingly, SDS did not leak out but stayed in the vesicle even after exhaustive dialysis in water and treatment with various external surfactants, due probably to the electrostatic effect within the vesicle. While spontaneous vesicles were also obtained at room temperature from cationic surfactants such as CTAB, DTAB, STAB, and DODAC in place of SDS, they were less stable during the polymerization, which required heating above 44 °C with VA044 and 60 °C with VA-060, undergoing rapid exclusion of the cationic surfactant from the vesicle to give a colloidal dispersion of a polymer micelle. On the other hand, replacement of SDS with other anionic surfactants such as SHS and SOA did not effect the loss of the surfactant but gave the corresponding polymerized vesicles. These results suggested that the electrostatic interaction within the vesicle between the anionic surfactant and the cationic VBTAC molecule was essential for surviving exposure to elevated temperatures required for the radical polymerization process. The polymerized vesicles were readily redispersed in water without the aid of external surfactants, by virtue of the presence of SDS incorporated in the particle. Figure 5 shows the SEM picture of the polymerized vesicle before the purification process, together with the DLS histogram obtained for the dispersion liquid. The polymerized vesicle was featured by the bimodal size distribution characteristics similar to that for the pristine vesicle in Figure 2. The mean Dh values were found at 290 and 1360 nm, which were roughly in agreement with the size of the primary particles estimated from SEM observations and almost doubled from those of the vesicles before the polymerization. The increase in the mean Dh indicated that some particles coalesced during the polymerization, which was suggested by a slight increase in the turbidity of the mixture. Nevertheless, the retention of the bimodal character of the size distribution indicated that intravesicular polymerization prevailed over intervesicular reaction under sufficiently diluted conditions. In control experi-
Figure 6. TEM picture of the polymerized vesicle after purification as in Figure 1c showing the hollow spherical structure.
ments, polymerization of the pristine vesicle without the 10-fold dilution in advance produced an insoluble precipitate rather than the dispersion, suggesting that the reaction was paralleled by the intervesicular polymerization under the concentrated conditions. The intravesicular polymerization of the vesicle allowed fixation of the hollow spherical structure. Figure 6 shows the TEM picture of the replica of the fractured vesicle after purification, which is spherical with a diameter of around 250 nm. The TEM image of the polymerized vesicle is quite similar, except in size, to that of the unpolymerized vesicles in Figure 2, indicating that the hollow structure is maintained during the polymerization. The hollow structure is supported by preliminary rheometry experiments as follows. The onset of an increase in the dispersion viscosity at high shear rates was observed at particle volume fractions as low as 3% for the polymerized vesicle, due to the presence of a large inner void and thus to the bulky structure of the particle. The onset value is lower than those for polystyrene particles with diameters of ca. 320 nm under the same experimental conditions (ca. 30%), and even lower than those for the corresponding hollow microspheres (ca. 10%) with diameters of ca. 1000 nm and a void volume of ca. 50%. It seems reasonable to suppose that the mixed micelles that coexist in the equilibrated mixture before polymerization are removed during the purification process of the polymerized vesicles. Intravesicular polymerization at an equilibrated state afforded a convenient method to provide a hollow nanosphere from the abundant amphiphiles. Our preliminary investigation revealed that the polymerized vesicles might be applied as a paper-coating material by taking advantage of remarkable visible light-scattering properties and the high viscosity of the hollow sphere dispersions, which is the topic of our continuous investigations.
Highly Stable Polymerizable Vesicles
Conclusions A VBTAC/SDS/DVB mixture gives highly stable spontaneous vesicles, probably equilibrated with a small amount of mixed micelles at suitable compositions. Polymerization of the vesicle is accomplished using external initiators. The method requires only the preparation of mixtures and polymerization with watersoluble radical initiators such as VA-044. Under suitable conditions, SDS and VBTAC behaved as an ion-paired unsaturated amphiphile to undergo copolymerization with DVB. The polymerization under diluted conditions took place intravesicularly, resulting in the retention of the bimodal size distribution
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characteristics of the pristine dispersion. SDS was incorporated in the polymerized vesicle due to the electrostatic interaction with the cationic cross-linked framework produced from VBTAC and DVB. The product is a hollow nanosphere with resistance to a wide variety of organic solvents and readily redispersed in water to give a pale blue colloidal dispersion liquid. Acknowledgment. This work was partially supported by Grants-in-Aid for Scientific Research (Nos. 16550128 and 17550138) from MEXT, Japan. LA053369G
