Langmuir 2002, 18, 6709-6711
Layer-by-Layer Self-Assembling of Liposomal Nanohybrid “Cerasome” on Substrates Kiyofumi Katagiri, Ryo Hamasaki, Katsuhiko Ariga, and Jun-ichi Kikuchi* Graduate School of Materials Science, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara 630-0101, Japan Received March 22, 2002. In Final Form: June 1, 2002
Introduction Advanced materials research on nanometer-scale architecture is one of the most dynamic realms of science and technology.1,2 The field is of fundamental significance, and a wide range of applications are possible. Lipid bilayer vesicles, so-called liposomes, are well-known nanomaterials which have been extensively employed as supramolecular assemblies to construct molecular devices.3-7 Recently we have demonstrated that an artificial signal transduction system can be constituted by functionalized liposomal assembly in combination with a bilayer-forming lipid, an artificial receptor, and an enzyme.8 In the next step, organization of such artificial cell models should be indispensable for the construction of supramolecular integrated circuits. Thus, it is of great interest to create a hierarchical multiliposomal assembly on a substrate. Up to the present time, however, there have been few reports on self-organization of liposomal particles.9-12 In particular, the multilayered piling of liposomes on a substrate and visualization of their morphology by atomic force microscopy has not been reported until now. The layer-by-layer assembly developed by Decher and co-workers is one of the most useful techniques to prepare multilayered ultrathin films on substrates. The method employs alternate adsorption of oppositely charged poly* Corresponding author. E-mail:
[email protected]. (1) Degani, R. Chem. Eng. News 2000, 78 (9), 36. (2) Balzani, V.; Credi, A.; Raymo, F. M.; Stoddart, J. F. Angew. Chem., Int. Ed. 2000, 39, 3348. (3) (a) Steinberg-Yfrach, G.; Liddell, P. A.; Hung, S.-C.; Moore, A. L.; Gust, D.; Moore, T. A. Nature 1997, 385, 239. (b) Steinberg-Yfrach, G.; Ligaud, J.-L.; Durantini, E. N.; Moore, A. L.; Gust, D.; Moore, T. A. Nature 1998, 392, 479. (4) Gokel, G. W.; De Wall, S. L. Redox control of aggregation in synthetic vesicles. In Advances in Supramolecular Chemistry; Gokel, G. W., Ed.; JAI Press: Stamford, CT, 1999; Vol. 5, p 203. (5) (a) Westmark, P. R.; Gardiner, S. J.; Smith, B. D. J. Am. Chem. Soc. 1996, 118, 11093. (b) Boon, J. M.; Smith, B. D. J. Am. Chem. Soc. 1999, 121, 11924. (c) Boon, J. M.; Smith, B. D. J. Am. Chem. Soc. 2001, 123, 6221. (d) Vandenburg, Y. R.; Zhang, Z.-Y.; Fishkind, D. J.; Smith, B. D. Chem. Commun. 2000, 149. (6) Lasic, D. D. Liposomes: from physics to applications; Elsevier: Amsterdam, 1993. (7) Gregoriadis, G., Ed. Liposome Technology; CRC Press: Boca Raton, FL, 1993; Vols. 1-3. (8) (a) Kikuchi, J.; Ariga, K.; Miyazaki, T.; Ikeda, K. Chem. Lett. 1999, 253. (b) Kikuchi, J.; Ariga, K.; Ikeda, K. Chem. Commun. 1999, 547. (c) Fukuda, K.; Sasaki, Y.; Ariga, K.; Kikuchi, J. J. Mol. Catal. B 2001, 11, 971. (d) Kikuchi, J.; Ariga, K.; Sasaki, Y.; Ikeda, K. J. Mol. Catal. B 2001, 11, 977. (9) Kim, S.; Turker, M. S.; Chi, E. Y.; Sela, S.; Martin, G. M. Biochim. Biophys. Acta 1983, 728, 339. (10) (a) Walker, S. A.; Kennedy, M. T.; Zasadzinski, J. A. Nature 1997, 387, 61. (b) Kisak, E. T.; Coldren, B.; Zasadzinski, J. A. Langmuir 2002, 18, 284. (11) Kogiso, M.; Ohnishi, S.; Yase, K.; Masuda, M.; Shimizu, T. Langmuir 1998, 14, 4978. (12) Patolsky, F.; Lichtenstein, A.; Willner, I. J. Am. Chem. Soc. 2001, 123, 5194.
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electrolytes and/or colloidal particles.13-17 The film growth can be performed in a cyclic manner, which is made possible by the overcompensation of surface charges when high molecular weight species are adsorbed at a solidliquid interface. A similar method has also been applied to liposome-forming lipids to prepare the supported lipid bilayers, but not to prepare the multiliposomal assembly.18-20 In general, liposomes change their aggregate morphology by collapse and fusion when they interact with polyelectrolytes with the opposite charge. Accordingly, a drastic increase in the morphological stability of liposomes seems to be more desirable for preparation of the multiliposomal assembly for such purpose. On these grounds we have recently designed a novel class of liposomes that are sufficiently stable to withstand multiliposomal assembly, keeping the original vesicular shape. We developed “Cerasome,” which forms a liposomal bilayer with a silicate framework on its surface, by adopting the sol-gel process for an organoalkoxysilane having a lipid-like structure.21-23 In this paper, we report for the first time that a multiliposomal assembly of Cerasome on inorganic substrates can be prepared by using the layer-by-layer assembling technique (Figure 1). Experimental Section Materials. The synthesis of N,N-dihexadecyl-N′-(3-triethoxysilyl)propylsuccinamide (1) was described in our previous paper.21 Dihexadecyl phosphate (DHP) was obtained from Aldrich Chemical Co., Inc. Polyelectrolytes, potassium poly(vinyl sulfate) (PVS), and poly(diallyldimethylammonium chloride) (PDDA) were purchased from Wako Pure Chemical Industries. 2-[4-(2Hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) used to prepare buffer solution was obtained from Dojindo Labora(13) (a) Decher, G.; Hong, J.-D. Ber. Bunsen-Ges. Phys. Chem. 1991, 95, 1430. (b) Decher, G.; Hong, J.-D.; Schmitt, J. Thin Solid Films 1992, 210/211, 831. (c) Lvov, Y.; Decher, G.; Mo¨hwald, H. Langmuir 1993, 9, 481. (d) Decher, G. Layered Nanoarchitectures via Directed Assembly of Anionic and Cationic Molecules. In Comprehensive Supramolecular Chemistry; Sauvage, J.-P., Hosseini, M. W., Eds.; Pergamon Press: Oxford, 1996; Vol. 9, p 507. (14) Bertand, P.; Jonas, A.; Laschewsky, A.; Legras, R. Macromol. Rapid Commun. 2000, 21, 319. (15) (a) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3422. (b) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3427. (c) Fery, A.; Scho¨ler, B.; Cassagneau, T.; Caruso, F. Langmuir 2001, 17, 3779. (d) Caruso, F. In Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mo¨hwald, H., Eds.; Marcel Dekker, Inc.: New York, 1999; p 193. (16) (a) Kotov, N. A.; Dekany, I.; Fendler, J. H. J. Phys. Chem. 1995, 99, 13065. (b) Kotov, N. A.; Haraszti, T.; Turi, L.; Zavala, G.; Geer, R. E.; Dekany, I.; Fendler, J. H. J. Am. Chem. Soc. 1997, 119, 6821. (c) Mamedov, A.; Ostrander, J.; Aliev, F.; Kotov, N. A. Langmuir 2000, 16, 3941. (d) Ostrander, J. W.; Mamedov, A. A.; Kotov, N. A. J. Am. Chem. Soc. 2001, 123, 1101. (17) (a) Lvov, Y.; Ariga, K.; Onda, M.; Ichinose, I.; Kunitake, T. Langmuir 1997, 13, 6195. (b) Onda, M.; Lvov, Y.; Ariga, K.; Kunitake, T. Jpn. J. Appl. Phys. 1997, 36, L1608. (c) Ichinose, I.; Tagawa, H.; Mizuki, S.; Lvov, Y.; Kunitake, T. Langmuir 1998, 14, 187. (d) Lvov, Y.; Munge, B.; Giraldo, O.; Ichinose, I.; Suib, S. L.; Rusling, J. F. Langmuir 2000, 16, 8850. (18) Ichinose, I.; Fujiyoshi, K.; Mizuki, S.; Lvov, Y.; Kunitake, T. Chem. Lett. 1996, 257. (19) (a) Zhang, L.: Longo, M. L.; Stroeve, P. Langmuir 2000, 16, 5093. (b) Zhang, L.; Booth, C. A.; Stroeve, P. J. Colloid Interface Sci. 2000, 228, 82. (20) Cassier, T.; Sinner, A.; Offenha¨user, A.; Mo¨hwald, H. Colloids Surf. B 1999, 15, 215. (21) Katagiri, K.; Ariga, K.; Kikuchi, J. Chem. Lett. 1999, 661. (22) Katagiri, K.; Ariga, K.; Kikuchi, J. Kobunshi Ronbunshu 2000, 57, 251. (23) Katagiri, K.; Ariga, K.; Kikuchi, J. In Studies in Surface Science and Catalysis; Iwasawa, Y., Oyama, N., Kunieda, H., Eds.; Elsevier Science: Amsterdam, 2001; Vol. 132; p 599.
10.1021/la025772i CCC: $22.00 © 2002 American Chemical Society Published on Web 07/20/2002
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Figure 1. Schematic drawing of three-dimensional selfassembled structure of the Cerasome prepared by the layerby-layer adsorption method.
Notes
Figure 2. Frequency decrease (-∆F) of QCM upon layer-bylayer assembling of anionic vesicle (0.5 mmol dm-3) and PDDA (open symbols, vesicle adsorption step; filled symbols, polyelectrolyte adsorption step): (a) Cerasome-PDDA (circles); (b) DHP-liposome-PDDA (triangles). Atomic Force Microscopy. To clarify the structure and morphology of the alternate layer-by-layer assembly, the multilayer films were examined by atomic force microscopy (AFM). AFM was conducted using a SPI-3800N System from Seiko Instruments Inc., Japan, in tapping mode with a 20 µm scanner. For AFM observations, a mica plate was chosen as a substrate for the alternate layer-by-layer assembling and used immediately after cleavage in a clean atmosphere.
tories. Water for the preparation and layer-by-layer assembling of Cerasome was distilled and deionized using an Autostill WS33 (Yamato Scientific) and Milli-Q Labo (Nihon Millipore), respectively. Standard solutions of diluted hydrochloric acid (0.1 mol dm-3 HCl; Wako Pure Chemical Industries) and aqueous ammonium hydroxide (28% NH4OH; Wako Pure Chemical Industries) were employed for pH control. Preparation of Cerasome and DHP-Liposome. An organoalkoxysilane (1) was used for preparation of aqueous dispersions of Cerasome by a manner in accordance with previous reports.21 Vortex mixing of 1 with aqueous HCl at pH 3 afforded a translucent vesicular solution, and then the pH of the solution was adjusted to 9.0 by adding aqueous NH4OH. Transmission electron microscopy and dynamic light scattering measurements revealed that the Cerasomes became multilayered vesicular structures with diameters of 50-300 nm. The isoelectric point as evaluated by ζ-potential measurements was 4.3, and the ζ-potential value was -60 mV at pH 9.0. The traditional anionic liposome derived from DHP dispersed in HEPES buffer (10 mmol dm-3, pH 7.0) was also prepared by vortex mixing. The DHPliposomes showed the same particle size and surface charge as Cerasome under the present experimental conditions: vesicular diameter, 50-300 nm; ζ-potential, -60 mV. Alternate Layer-by-Layer Assembling. A well-defined precursor film of anionic PVS and cationic PDDA was assembled onto the resonator. Aqueous solutions of PVS and PDDA were prepared by using HEPES buffer (10 mmol dm-3, pH 7.0) at concentrations of 4 and 6 mg mL-1, respectively. The precursor film contained five polyelectrolyte layers beginning with PDDA in the alternate mode of PDDA/PVS, and the terminal layer was positive PDDA. A substrate was then alternately immersed for 20 min in an aqueous dispersion of the liposome (Cerasome or DHP-liposome) and aqueous PDDA with intermediate water washing and drying under a nitrogen stream. Quartz Crystal Microbalance Measurement. The quartz crystal microbalance (QCM) from USI System Co. was used for detection of mass changes during the layer-by-layer assembling process. The QCM resonator was covered by vapor-deposited silver electrode on both faces, and the resonance frequency was 9 MHz (AT cut). The QCM frequency decreases (-∆F) proportionally upon mass increase.24-27 All experiments were conducted at pH 9.0 and 25 °C. (24) Sauerbery, G. Z. Phys. 1959, 155, 206. (25) Caruso, F.; Serizawa, T.; Furlong, D. N.; Okahata, Y. Langmuir 1995, 11, 1546 (26) Lvov, Y. In Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mo¨hwald, H., Eds.; Marcel Dekker: New York, 2000; p 125.
Results and Discussion Alternation with the oppositely charged polycations is essential for successful deposition of the anionic Cerasomes. In addition, the QCM frequency decrease value did not change when the resonator was washed repeatedly, suggesting that Cerasomes were adsorbed tightly on the cationic PDDA layer. On the other hand, the negatively charged Cerasomes were not adsorbed onto the anionic PVS surface. The frequency changes observed at each step in the assembly of the Cerasome in alternation with PDDA are shown in Figure 2. The odd-numbered steps correspond to Cerasome adsorption, and the even-numbered steps correspond to PDDA adsorption. For comparison, the frequency decreases observed for the assembly of traditional anionic liposome derived from DHP dispersed in HEPES buffer in alternation with the cationic PDDA are also given in Figure 2. Regular film growth was found in all cases. Larger and smaller frequency changes correspond to adsorption of the liposomes and PDDA, respectively. In addition, the adsorbed mass was much greater for the Cerasome layer than for the layer for DHPliposome. The average -∆F value for a single Cerasome adsorption step was 1700 Hz, and that for DHP-liposome was 360 Hz. In our system, the following relationship is obtained between adsorbed mass, W (g), and frequency change, ∆F (Hz), by taking into account the characteristics of the quartz resonator
∆F ) (1.83 × 108)W/A
(1)
where A is the cross-sectional area of the quartz microbalance placed between QCM electrodes and is 0.16 ( 0.01 cm2. Then, by using the limiting molecular area of the lipid molecule, A′ (0.4 nm2), and the molecular weights of DHP and the Cerasome-forming lipid molecule, M (546.8 and 685.2, respectively), the adsorbed mass for planar bilayer is obtained from the following relationship: (27) (a) Ebara, Y.; Okahata, Y. J. Am. Chem. Soc. 1994, 116, 11209. (b) Ebara, Y.; Itakura, K.; Okahata, Y. Langmuir 1996, 12, 5165.
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W ) 2(2A/A′)/(6.02 × 1023)M
(2)
On the other hand, the adsorbed mass for a layered assembly with spherical liposomal particles is calculated from the following relationships:
W ) F(4/3)(πr3)Na
(3)
Na ) (0.9069 × 2A)/πr2
(4)
where Na is the number of liposomes adsorbed on both sides of the resonator, and r is the supposed radius of the liposomes (50 nm). The coefficient of two-dimensional packing, 0.9069, and the supposed density of liposome (F) are also used in this calculation. If ideal hexagonal packing with 100% coverage is attained for a planar bilayer of DHP and for the Cerasome-forming lipid, the QCM resonator will give frequency decreases of 170 and 210 Hz, respectively. On the other hand, if the hexagonal packed layer of liposomal particles in a diameter of 100 nm is obtained, the QCM resonator will give a frequency decrease of 2130 Hz with 100% coverage. In the case of DHP-liposome, the -∆F value observed experimentally was roughly twice as much as the value calculated for a planar-supported bilayer. It suggests that the DHPliposomes were mainly deposited as a planar bilayer membrane after the PDDA-induced fusion during the adsorption process. In contrast, the -∆F value for the Cerasome adsorption was too large for the planarsupported bilayer deposition of the lipid and corresponded to the value calculated for the layered assembly of spherical vesicular particles with coverage of ca. 75%. That is, the Cerasomes were assembled on the substrate without collapse and membrane fusion during the adsorption process. An AFM image of the PVS-PDDA precursor film on a mica substrate is shown in Figure 3a. A relatively flat surface with a height difference of 1 nm over 600 nm in the horizontal distance was observed from a height profile of this image. This is consistent with the findings reported for the sodium poly(styrenesulfonate)-PDDA assembly.17b In contrast, the Cerasome particles were clearly seen in the AFM image of the Cerasome-PDDA film on a mica plate for the first adsorption step of the vesicle (Figure 3b). The Cerasome particles are closely packed like a stone pavement in the layer. Such morphology was found in the whole area of the mica substrate. We found similar morphology for the second, third, and more adsorption steps of the Cerasome layer in the AFM images (an image obtained after the fifth adsorption step is shown in Figure 3c). On the other hand, the stone-paving morphology could not be observed for the DHP-liposome-PDDA assembly (Figure 3d), an observation that is consistent with the reported results to prepare the supported bilayer on a substrate.19 In this case, DHP liposomes collapsed and induced fusion through electrostatic interactions with PDDA, since the vesicular morphology was not so stable. Thus we can obtain three-dimensional layered organization of nanoparticles with vesicular structure only for the Cerasome having a stable inorganic framework on the membrane surface. Although there are a few reports for immobilization and direct visualization of liposomes on
Figure 3. Tapping mode AFM images of the surface of the alternate layer-by-layer assembled films on the mica substrate: (a) PVS-PDDA assembled precursor film; (b) Cerasome-PDDA assembled film (after first adsorption step shown in Figure 2a); (c) Cerasome-PDDA assembled film (after fifth adsorption step shown in Figure 2a); (d) DHP-liposome-PDDA assembled film (after first adsorption step shown in Figure 2b).
the substrates as observed by AFM,28,29 these samples were not prepared by the alternate layer-by-layer assembling method and the liposomes were randomly deposited on the substrate and not closely packed. Therefore, it is notable that the well-packed paving morphology of the Cerasome is observed in each layer despite the strong electrostatic interactions between the Cerasome and PDDA. Conclusions In conclusion, a three-dimensionally packed liposomal assembly on inorganic substrates was successfully prepared by using an organic-inorganic hybrid vesicle, the Cerasome. To the best of our knowledge, this is the first example of construction and direct visualization of the three-dimensional liposomal assembly prepared by using the layer-by-layer adsorption method. These organized Cerasomes have potential as advanced nanomaterials exhibiting artificial multicellular functions. Acknowledgment. This work was supported by the Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), 12450364, 2001. LA025772I (28) Kumar, S.; Hoh, J. H. Langmuir 2000, 16, 9936. (29) Stanish, I.; Santos, J. P.; Singh, A. J. Am. Chem. Soc. 2001, 123, 1008.
