© Copyright 2001 American Chemical Society
APRIL 17, 2001 VOLUME 17, NUMBER 8
Letters Formation of Multilayer Composite Particles Comprised of Silica/Vesicle/Silica Particles by Heterocoagulation Bo Yang,† Hideo Matsumura,‡ Kaoru Katoh,‡ Hideo Kise,† and Kunio Furusawa*,§ Institute of Material Science, University of Tsukuba, Tsukuba, Ibaraki 305, Japan, Electrotechanical Laboratory, AIST, MITI, Tsukuba, Ibaraki 305, Japan, Department of Chemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan Received July 31, 2000. In Final Form: December 12, 2000 The multilayer composite particles comprised of silica/PC (phosphatidylcholine) vesicle/silica particles have been prepared by using the heterocoagulation technique. The formation process is as follows: at first, large silica particles (2r ) 1.5 µm) and PC vesicles (2r ) 0.2 µm) were mixed at a definite particle number ratio in 10-4 M LaCl3 aqueous solution. The PC vesicle/silica composites were spontaneously formed by mutual electrostatic attraction. After removal of the excess PC vesicles from the system, the small silica particle (2r ) 0.5 µm) suspension was added into the PC vesicle/silica composite dispersions to form the multilayer composite particles composed of small silica, PC vesicle, and core silica particles. The formation of multilayer composite particles was confirmed by the electrophoresis, dynamic light scattering (DLS), and adsorption measurements of PC molecules. Finally, the existence of multilayers was confirmed directly using a special optical microscope.
1. Introduction So far, the studies of composite particles have focused on the organic-inorganic composite particles, such as latex-silica,1-5 latex-alumina,6 latex- or silica-magnetic particle,7-10 and so on. Further, according to the recent intensive studies, there are a variety of methods currently * To whom correspondence should be addressed. Telephone and Fax: 81 298 53 4426. E-mail:
[email protected]. † Institute of Material Science, University of Tsukuba. ‡ AIST. § Department of Chemistry, University of Tsukuba. (1) Harding, R. D. J. Colloid Interface. Sci. 1972, 40, 57. (2) (a) Furusawa, K.; Anzai, C. J. Colloids Surf., A 1992, 63, 103. (b) Furusawa, K.; Nagashima, K.; Anzai, C. J. Colloid Polym Sci. 1994, 272, 1104. (c) Furusawa, K.; Kimura, Y.; Tagawa, T. J. Colloid Interface Sci. 1986, 109, 69. (3) Honda, H.; Kimura, M.; Honda, F.; Matsuno, T.; Konishi, M. J. Colloids Surf., A 1994, 82, 117. (4) Otsubo, Y.; Kazaya, E. J. Colloid Interface Sci. 1994, 168, 230. (5) Caruso, F.; Mohwald, H. Langmuir 1999, 15, 8276. (6) Esmui, K.; Watanabe, N.; Meguro, K. Langmuir 1991, 7, 1775. (7) Kemshead, J. T.; Ugelstad, J. Mol. Cell. Biochem. 1985, 67, 11.
used to fabricate a wide range of stable hallow spheres of various compositions by removing the emulsion droplets or latex particles used as templates.11-14 However, composite formations including soft biocolloids (e.g. vesicle) have not been reported. As is generally known, vesicles are widely studied and used as carriers of some medicines (8) Pieters, B. R.; Williams, R. A.; Webb, C. Magnetic Carrier Technology. In Colloid and Surface Engineering: Application in the Process Industries; Williams, R. A., Ed.; Butterworth: Oxford, 1992. (9) Kawahashi, N.; Matijevic, E. J. Colloid Interface. Sci. 1990, 138, 534. (10) Philipse, A. P.; van Bruggen, M. P. B.; Pathmamanoharan, C. Langmuir 1994, 10, 92. (11) (a) Velev, O. D.; Furusawa, K.; Nagayama, K. Langmuir 1996, 12, 2374. (b) Velev, O. D.; Furusawa, K.; Nagayama, K. Langmuir 1996, 12, 2385. (12) Schacht, S.; Huo, Q.; Voigt-Martin, I. G.; Stucky, G. D.; Schuth, F. Science 1996, 273, 768. (13) Bamnolker, H.; Nitzan, B.; Gura, S.; Margel, S. J. Mater. Sci. Lett. 1997, 16, 1412. (14) Caruso, F.; Caruso, R A.; Mohwald, H. Science 1998, 282, 1111.
10.1021/la001088x CCC: $20.00 © 2001 American Chemical Society Published on Web 03/16/2001
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Figure 1. Schematic showing the process of synthesizing of the multilayer composite particles: (a) core silica; (b) PC vesicle/silica composite particle; (c) silica/PC vesicle/silica multilayer composite particle.
in drug delivery systems,15-18 or imaging agents,19 and in the cosmetic industry, due to their advantages: reduced toxicity at constant efficacy, and biocompatibility. So, the composite particles including vesicle particles may be used successfully in biomedicine and the cosmetic industry. Furthermore, by our recent studies on the adsorption behavior of PC vesicles at air/water or oil /water20,21 and solid/water interfaces,22 it is confirmed that the vesicles may keep their spherical shape at these interfaces. Especially, at the solid/water interface, the vesicle formation is stable. All these results suggest that PC vesicles can be used as a component of the multilayer composite particles. In this study, vesicles/silica and further silica/ vesicle/silica composite particles were constructed in a stepwise fashion by using the heterocoagulation technique in a 10-4 M LaCl3 aqueous solution. 2. Experimental Section Materials. Egg yolk phosphatidylcholine (PC) was purchased from Sigma Chemical Co., Ltd. (USA). The silica particles (two sizes: 1.5 µm and 0.5 µm diameter) were supplied by Nippon Catalysis Co., Ltd. (Japan). Prior to being used, the powder samples of silica particles were dispersed in a flask with pure water by sonication for 20 min. Inorganic chemicals were of analytical reagent grade and were supplied by Wako Pure Chemical Industry (Japan). The water used in all experiments was purified by the Nanopure system and redistilled in a Pyrex model still-1 (Iwaki Glass Co., Ltd., Japan). Preparation of Vesicles. Unilamellar PC vesicles were prepared by the extrusion method according to Hope et al.23 First, 30 mg of the PC lipid was dissolved in CHCl3. The solution was evaporated to form a lipid film on the flask wall. Thereafter, pure water (10 mL) was added, and the mixture was sonicated to obtain a multilayer large vesicle (MLV) suspension. After five freeze/thaw cycles, the lipid dispersion was extruded five times through two stacked polycarbonate filters. The mean diameter of the resulting vesicles was determined with a light scattering apparatus (Otsuka Elect. ELS-800). In this study, the PC vesicle
with 0.2 µm diameter was usually used. The PC concentration in the vesicle suspension was determined by the Bartlett method.24 Electrophoresis. The electrophoretic mobility of the silica particles, vesicles, and composite particles was measured in 10-4 M LaCl3 aqueous solution by using a microelectrophoretic apparatus (Zeecom; Microtech Nichion. Co., Ltd., Japan). The ζ-potentials were calculated from the mobility data using the O’Brien-White equation.25 Dynamic Light Scattering (DLS). The mean sizes of the vesicles and composite particles were analyzed using a dynamic light scattering apparatus (ELS-800; Otsuka Elect. Co., Ltd., Japan). Amount of PC Adsorption. The dispersion of 1 mL of large silica (2r ) 1.5 µm), which included 5 mg of silica particles, was added to 1 mL of PC vesicle dispersion, and the mixture was stirred for 4 h at 25 °C. The sample was centrifuged at 800g for 30 min at 15 °C to separate the supernatant particles from the vesicles in the bulk. The supernatant and the original vesicle dispersions were used to determine the phospholipid concentration by the Bartlett method.24 The PC adsorption amount on the silica particles was calculated from these data. Optical Microscopy. The prepared composite particles were imaged using a video-enhanced differential interference contrast microscope (VEDIC). The following is a list of instrumental parts and settings used in the present study. The microscope is equipped with an oil immersion condenser lens with an aperture diaphragm (maximum numerical aperture of 1.4, typically set to 1.0) and a 60X/1.4 numerical aperture plan apochromat objective lens, both selected for low-polarization aberrations. A mercury arc lamp followed by an Ellis light scrambler (Technical Video, Woods Hole, MA) was used to homogeneously illuminate the back aperture of the condenser, and narrow band-pass interference filters (546 nm, 10 nm fwhm, Omega Optical Brattleboro, VT) were used to select the green mercury line for monochromatic illumination. A video camera (CCD-300-RC, Dage-MTI Inc, Michigan City, IN) is attached to the camera port. A computer with a frame grabber board (AG-5, Scion Co., MD) to capture the images, and a z-axis controller (Flovel Co., Tokyo) to record the z-series (typically step size, 50 nm) of the images were used.26
3. Results and Discussion (15) Bangham, A. D., Ed. Liposomes Letters; Academic Press: New York, 1983. (16) Gregoriadis, G., Ed. Liposomes Technology; CRC Press: Boca Raton, FL, 1992. (17) Lasic, D. D. Liposomes: Form Physics to Applications; Elsevier: Amsterdam, 1993. (18) Maruyama, K.; Unezaki, S.; Takahashi, N.; Iwatsuru, M. Biochim. Biophys. Acta 1993, 1149, 209. (19) Seltzer, S. E.; Janott, A. S.; Blau, M.; Adams, D. F.; Minchey, S. R.; Boni, L. T. Invest. Radiol. 1991, 26, 169. (20) Yang, B.; Matsumura, H.; Furusawa, K. Colloids Surf., B 1999, 14, 161-168. (21) Yang, B.; Matsumura, H.; Kise, H.; Furusawa, K. Langmuir 2000, 16, 3160-3164. (22) Yang, B.; Matsumura, H.; Kise, H.; Furusawa, K. Stud. Surf. Sci. Catal. 2001, 132, 415-418. (23) Hope, M. J.; Bally, M. B.; Webb, G.; Cullis, P. K. Biochim. Biophys. Acta 1985, 812, 55.
3-1. Preparation of Composite Particles. The silica/ PC vesicle/silica multilayer composite particles were prepared by the alternate adsorption of PC vesicles and small silica particles on the large silica particles. The schematic picture showing the fabrication process is indicated in Figure 1. Here, the electrostatic attraction between the component particles is taken into account as a driving force. So, it is of importance to control the surface potentials of silica and vesicles particles to opposite sign. (24) Bartlett, G. R. J. Biol. Chem. 1959, 234, 466. (25) O’Brien, R. W.; White, L. R. J. Chem. Soc., Faraday Trans. 1978, 74, 1607. (26) Inoue S.; Spring, K. R. Video Microscopy, 2nd ed.; Plenum Press: New York, 1997.
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Table 1. Diameter and ξ-Potential of Various Particles in 10-4 M LaCl3 Aqueous Solution particles or composite PC vesicle core silica particles shell silica particles PC vesicle/core silica composite small silica/PC vesicle/core silica composite a
diameter (µm) ξ-potential (mV) 0.2a 1.5a 0.5a 1.93a (1.9b) 2.8c (2.9b)
+32.0 -30.2 -30.2 +28.4 -30.0
By DLS. b By calculation. c By optical microscopy.
Figure 2. Adsorption amount of PC on core silica surface in10-4 M LaCl3 at 25 °C.
Figure 4. Optical micrographs of silica/PC vesicle/silica multilayer composite particles: (a) top level; (b) middle level.
Figure 3. Optical micrographs of core and shell silica particles.
According to the ζ-potential measurements characterizing the component particles, these particles show different affinities for La3+ ion and the ζ-potentials of silica and PC vesicles were -30.0 mV and +32.0 mV at 10-4 M LaCl3. So, we expect that a selective electrostatic attraction between the vesicles and silica particles will operate in 10-4 M LaCl3 aqueous solution. The actual formation process is as follows. The dispersions of silica particles and PC vesicles were prepared separately in a 10-4 M LaCl3 aqueous solution (the particle concentrations are 2.5 × 109/mL and 1.2 × 1012/mL, respectively, for silica and PC vesicles). Equal volumes of two dispersions were mixed using a rapid mixing apparatus (MX-7, Union Giken. Co., Ltd., Japan), with a jet device for rapid mixing and control. The mixture dispersion was then continuously stirred for 2 h at room temperature for stabilization of the PC vesicle/silica composite particles. Subsequently, the excess of PC vesicles that remained in the suspension was removed by filtration (pore size ) 1.0 µm filter), and the remains were washed three times with a 10-4 M LaCl3
aqueous solution. After that, the resulting composite dispersion and the silica dispersion including a small sized silica (2r ) 0.5 µm) were mixed by the same procedure and under the same conditions as in the first adsorption step. After standing overnight, the multilayer composite particles and excess of small silica particles were separated by spontaneous precipitation. By this way, it is possible to obtain stable multilayer composite particles. 3-2. Confirmation of Multilayer Structure. The formation of composite silica/PC vesicle/silica particles has been supported by several experimental data points. First, the composite formation of PC vesicle/silica particles was supported briefly by the ζ-potential and DLS measurements of the samples. As seen from Table 1, the ζ-potentials of the original silica and PC vesicles are -30.2 mV and +32.0 mV, respectively, at 10-4 M LaCl3. Therefore, it is expected that a strong electrostatic attraction may operate between these particles, and stable composite particles are formed. As given in Table 1, the coagulation products from these particles showed a value of z ) +28.4 mV, which may be due to the adsorption of vesicles on the adsorbent of the silica surface. Furthermore, it was found that multilayer products showed a negative value of the ζ-potential in comparison to that for the original silica particles. It seems likely that DLS data for the products support our consideration of a clear
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adsorption mechanism, where the average determined diameter of the composite particles is 1.93 µm. This is very close to the value of 1.9 µm calculated from the singlelayer model of vesicle particles (Figure 1b). Second, as relatively powerful evidence with which to prove the formation of a PC vesicle layer on a silica surface, the adsorption amount of PC vesicles was introduced. The adsorption process of PC molecules showed two possible kinds of adsorption. The first is the formation of a single layer of vesicle particles, and the second is the formation of a PC molecular bilayer. They both give similar positive ζ-potentials. However, the amounts of adsorption were different in each case. Figure 2 shows the adsorption amounts of PC molecules as a function of the PC concentration. Here, the adsorption amounts are expressed by the number of PC molecules adsorbed per square meter of silica surface. The solid line represents a calculated curve for the adsorption amount of the PC molecule bilayer model assuming that the area per PC molecule equals 0.7 nm2 (the value is 25 × 1017 PC molecules/m2). The dotted line shows a calculated curve for the adsorption of a singlelayer model of vesicle particles in the case that the vesicles are of uniform size particles having unilamellar spherical shape (the value is 160 × 1017 PC molecules/m2). Indeed, the saturated adsorption amounts determined experimentally (about 200 × 1017 PC molecules/m2) approached the dotted line and finally exceeded it. Furthermore, if we assume a multi-bilayer adsorption of PC molecules, the amount of PC corresponding adsorption (Figure 2) is approximately eight times larger in comparison to that for a single bilayer. Such a diameter of a composite particle can attain only 1540 nm (i.e., 1500 nm + 5 × 8 nm, because the single bilayer thickness is about 5 nm), which is fairly different from the 1930 nm value obtained from the DLS
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experiments. All these results suggest that PC vesicles adsorb on a silica surface as a single layer of vesicle particle, and the composite particles are generated according to the scheme in Figure 1. Finally, the direct observation of the multilayer formation of composite particles is provided by using a special optical microscope. In Figure 3, as a control, a photo for a mixture of core silica particles and small silica particles in the 10-4 M LaCl3 solution without PC vesicles is shown. We can see that they are well dispersed as a single particle. In Figure 4, photos of silica/PC vesicle/silica composite particles are indicated. Here, two photos are taken by changing the focusing positions of the optical microscope from the up side (a) to the middle (b) of a composite particle. All these photos clearly show that the small silica particles are adsorbed on the spherical surface of the PC vesicle/ core silica composite and are located on the outer layer of the composite particles. 4. Conclusions The silica/PC vesicle/silica multilayer composite particles were prepared by the alternative adsorption of PC vesicles and small silica particles on large silica particles in 10-4 M LaCl3 aqueous solution. The multilayer structure resulting was confirmed by the electrophoretic mobility, DLS measurements, and measurement of the adsorption amount of PC on a silica surface and by direct observation using a special optical microscope. Acknowledgment. Prof. Vassil Neytchev (Institute of Biophysics, Bulgarian Academy of Science) is acknowledged for making corrections to the manuscript. LA001088X
