Facile Method for Preparing Liposomes by Permeation of Lipid

Jul 11, 2013 - The effects of lipid concentration, pore size of the SPG membrane, and the alcohol used for the lipid solution on the structures and pr...
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Facile Method for Preparing Liposomes by Permeation of Lipid− Alcohol Solutions through Shirasu Porous Glass Membranes Kazuki Akamatsu,* Yusuke Shimizu, Ryutaro Shimizu, and Shin-ichi Nakao Department of Environmental and Energy Chemistry, Faculty of Engineering, Kogakuin University, 2665-1 Nakano-machi, Hachioji-shi, Tokyo 1920015, Japan ABSTRACT: We present a novel and facile approach for preparing monodispersed liposomes simply by permeating lipid− alcohol solutions through Shirasu porous glass (SPG) membranes. This method uses the phenomenon of mixing of two miscible liquids at their interface formed near an SPG membrane surface, which induces spontaneous formation of liposomes. The effects of lipid concentration, pore size of the SPG membrane, and the alcohol used for the lipid solution on the structures and properties of the liposomes are studied. The liposome size is around 120 nm, and the size distribution is narrow, regardless of the lipid concentration, pore size, and alcohol used. However, the alcohol used affects the property of liposomes: An increase in the liposome size is clearly observed in a freezing−thawing treatment when isopropyl alcohol is used as the alcohol, whereas the liposome size is unchanged by the freezing−thawing treatment when ethanol is used. sandwiched in the middle of the two PBS flows. At the interfaces between the IPA solution and PBS, IPA and water mix with each other by diffusion, which reduces the concentration of the IPA solution and facilitates fabrication of liposomes. Unlike other methods using microfluidic devices, this method is not based on emulsification for preparing liposomes; it just uses mixing of two miscible liquids at their interface, and accordingly induces liposome formation. Such an interface between two miscible liquids can also be formed using membranes, as shown in Figure 1b. This enables the preparation of liposomes by simply permeating lipid−alcohol solutions through membranes. Compared with the method using the microfluidic device, an increase in the interface is achieved in this method. Additionally, a further increase in the interface can easily be achieved by increasing the surface areas of the membranes, and membrane technology is well suited to such a scale-up. In this study, Shirasu porous glass (SPG) membranes are used. The pore-size distribution of the SPG membranes is narrow, so the membrane emulsification technique using SPG membranes is well-known as a sizecontrolling emulsification technique.15,16 There have been many articles reporting various functional particle synthesis methods using emulsions prepared using SPG membrane emulsification as a templates,17−24 although our method does not use emulsions. However, there has only been one article on the combination of liposomes and SPG membranes, focused on the narrow pore-size distribution of SPG membranes, by Hwang et al.25 They first prepared liposomes simply using a film-hydration method, and then repeatedly extruded the prepared liposome dispersion through SPG membranes under pressure. They successfully demonstrated that the size of the liposomes can be controlled by the passage times and pore sizes

1. INTRODUCTION Liposomes (lipid vesicles) are spherical compartments formed in vitro in aqueous media. They consist of lipid bilayer membranes, in which aqueous components can be encapsulated and sequestered. Because of their biocompatibility, liposomes have attracted much attention in the pharmaceutical and cosmetic fields. In recent years, there have been interesting publications focusing on novel functions induced on liposome membranes and demonstrating innovative functional materials by building up such liposome membranes systematically.1−3 Since Bangham and co-workers first developed liposomes,4 the simplest and most widely used method for preparing liposomes has been the thin-film hydration method. Because the liposomes prepared using this method are multilamellar and their size distribution is usually polydispersed, sonication and/ or extrusion methods are often used as postprocessing steps in order to obtain size-controlled and monodispersed unilamellar vesicles. As well as the thin-film hydration method, many other preparation methods have been established, such as alcohol injection,5,6 reverse-phase evaporation,7 and use of supercritical carbon dioxide.8,9 The liposome size, monodispersity, and lamellarity depend on the preparation method and preparation conditions. In the 2000s, various novel preparation methods using microfluidic devices have been developed. Takeuchi and co-workers10 and Fletcher and co-workers11 demonstrated high-throughput production of size-controlled liposomes by microfluidic jetting of planar lipid membranes. Weitz and coworkers12 and Ménager and co-workers13 developed monodispersed and size-controlled liposomes from W/O/W emulsions containing lipids in the oil phase, prepared using glass microcapillary devices. Jahn et al. also developed an interesting method for preparing vesicles using microfluidic channels.14 In their method, isopropyl alcohol (IPA) containing dissolved lipids flows through the center inlet channel, and phosphate-buffered saline (PBS) flows through the two side inlet channels, as shown in Figure 1a. At the cross-junction in the microfluidic device, a stream of the lipid−IPA solution is hydrodynamically © 2013 American Chemical Society

Received: Revised: Accepted: Published: 10329

June 13, 2013 July 8, 2013 July 11, 2013 July 11, 2013 dx.doi.org/10.1021/ie401876z | Ind. Eng. Chem. Res. 2013, 52, 10329−10332

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Figure 1. Conceptual diagrams of liposome formation (a) with a microfluidic device, studied by Jahn et al.,14 and (b) with an SPG membrane, studied by us.

are shown in Figure 2a,b. The samples were negatively stained with phosphotungstic acid. These TEM images show that the

of the SPG membranes. This is an interesting study, but the research concept is based on the extrusion method, and is different from ours. In this study, we use SPG membranes to prepare liposomes from lipid−alcohol (IPA or ethanol) solutions; however, this novel concept is not based on the emulsion-template method. This method aims at spontaneous formation just by mixing two miscible liquids at their interface formed near the SPG membrane surface.

2. EXPERIMENTAL SECTION Materials. 3-sn-Phosphatidylcholine (PC) from egg yolk, cholesterol, chloroform, isopropyl alcohol (IPA), ethanol, sodium chloride, potassium chloride, disodium hydrogen phosphate dodecahydrate, and sodium dihydrogen phosphate dehydrate were purchased from Wako Pure Chemical Industries Ltd., Osaka, Japan. All chemicals were used without further purification. Preparation of Lipid Thin Film. PC (50 mol %) and cholesterol (50 mol %) were dissolved in chloroform. After evaporation of the solvent using a rotary evaporator, the resultant thin film was dried under vacuum for 24 h to remove the solvent completely. Then the thin film was dissolved again in IPA or ethanol to yield 10 mM or 1 mM lipid solutions. Preparation and Characterization of Liposomes. An external-pressure-type microkit17 (SPG Techno Co. Ltd., Miyazaki, Japan) was used to prepare the liposomes. The SPG membranes used were hydrophilic, and had pore sizes of 1.1, 0.5, or 0.2 μm (tubular type, ϕ 1 cm, length 20 mm, SPG Techno Co., Ltd.). The lipid−alcohol solution was poured into a dispersion tank and then dispersed slowly through the SPG membrane into an aqueous solution (50 mL) containing sodium chloride, potassium chloride, disodium hydrogen phosphate, and sodium dihydrogen phosphate, without stirring. For stable preparation, the permeation rate of the lipid-alcohol solution through the SPG membranes was kept less than 6 g h−1. The resultant liposomes were characterized using dynamic light scattering (ELSZ-2KH, Otsuka Electronics Co., Ltd.) and transmission electron microscopy (JEM-100, JEOL Ltd.,). In the freezing-thawing tests, the liposome solution was frozen in liquid nitrogen and placed at room temperature. This freezing− thawing treatment was repeated five times to determine the properties of the liposomes.

Figure 2. TEM images of particles prepared using a 10 mM lipid solution and an SPG membrane with a pore size of 1.1 μm. The alcohol for the lipid solution is (a) IPA or (b) ethanol.

particles we prepared using the novel method are multilamellar vesicles, regardless of the solvent used. Figure 3a,b, respectively, show the size distribution of the liposomes when IPA or ethanol was used as the solvent. The average diameter of the liposomes in the case of IPA as the solvent is 121 nm, and that in the case of ethanol is 124 nm, indicating that the liposome diameter does not depend on the solvent. It should also be

3. RESULTS AND DISCUSSION Transmission electron microscopy (TEM) images of the particles prepared from a 10 mM lipid−IPA or lipid−ethanol solution, using an SPG membrane with a pore size of 1.1 μm, 10330

dx.doi.org/10.1021/ie401876z | Ind. Eng. Chem. Res. 2013, 52, 10329−10332

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Research Note

Figure 3. Size distributions of liposomes when (a) IPA or (b) ethanol was used as the solvent for the lipid.

Figure 4. Relationship between pore size of the SPG membrane and liposome size when the lipid concentration is fixed at 10 mM (open symbols) or 1 mM (closed symbols). The lipid solvent is (a) IPA or (b) ethanol.

noted that these diameters are much smaller than the pore size of the SPG membrane (1.1 μm). This is a result of the liposome formation mechanism. In the microfluidic jetting method,10,11 the liposome size depends on the size of the compartment blown out from the planar lipid membranes, therefore the liposome sizes are larger than 100 μm. In the W/ O/W-emulsion-template method,12,13 the liposome size depends on the size of the W/O/W emulsion prepared using a microfluidic device, which results in the formation of liposomes of size 20−150 μm. These liposomes are quite large. It is well-known that in the membrane emulsification technique using SPG membranes, the size of the emulsions is around three times larger than that of the pore size of the SPG membranes. However, this relationship between the emulsion size and the pore size only holds in the case of stable emulsification. In this study, we do not aim to prepare emulsions for fabricating liposomes. We simply use mixing a lipid−IPA solution or a lipid−ethanol solution with water at the interface formed near the SPG membrane surface, inducing spontaneous liposome formation. In fact, in the microfluidic method using the mixing of IPA with water, by Jahn et al.,14 the liposome size ranged from 100 to 300 nm, which is much smaller than those obtained using other methods, and is quite compatible with those obtained using our method. This common mechanism inducing liposome formation results in liposomes of a size around 120 nm in our method. On the basis of the discussion above, it is not unexpected that the liposome diameters are much smaller than the pore size of the SPG membrane. As for the monodispersity of the liposomes, δ for the IPA and ethanol solutions are 0.58 and 0.59, respectively; δ is defined as

pore size of the SPG membrane and lipid concentration, and ranges from 96 to 127 nm, even when the pore size ranges from 0.2 to 1.1 μm, and even when the lipid concentration ranges from 1 mM to 10 mM. These results indicate that the liposome size is much smaller than the pore size of the SPG membrane, regardless of the solvent used; this also demonstrates that this novel method does not use an emulsion template but involves the mixing of two miscible liquids at their interface near the SPG membrane surface for liposome preparation. Figure 5 panels a and b show the relationship between the number of freeze−thaw cycles and the average diameter for

Figure 5. Relationship between the number of freeze−thaw cycles and average diameter for liposomes prepared from a 10 mM lipid solution using an SPG membrane of pore size 1.1 μm. The lipid solvent is (a) IPA or (b) ethanol.

liposomes prepared from a 10 mM lipid solution using an SPG membrane of pore size 1.1 μm. The solvent for the lipid is IPA or ethanol. When the solution is frozen, the trapped aqueous volume increases due to ice formation, which disrupts liposome membranes. And when the solution is thawed, the membranes refused and the diameters of the liposomes increased. This freeze−thaw cycle is often used for drug-loading, and at the same time we can investigate the liposome properties, in particular the membrane fluidity. When IPA is used for preparation of the liposomes, the average diameter gradually increases with the number of freeze−thaw cycles, and finally reaches 339 nm. In contrast, when ethanol is used for preparation of the liposomes, the average diameter does not increase during the cycles. It is interesting that the alcohol affects the liposome properties, in particular the membrane fluidity, although the alcohol does not affect the liposome

δ = (D90 − D10)/D50

where D10, D50, and D90 are the cumulative number percentage of particles with diameters up to D10, D50, and D90, respectively. Very highly monodispersed particles have a δ of around 0.4,26 but our values are quite good. Figure 4 panels a and b show the relationship between the pore size of the SPG membrane and liposome size when the lipid concentration is fixed at 10 mM or 1 mM. The solvent for the lipid is IPA or ethanol. When IPA is used as the solvent, the liposome size hardly depends on the pore size of the SPG membrane and lipid concentration, and ranges from 97 to 151 nm, even when the pore size ranges from 0.2 to 1.1 μm, and even when the lipid concentration ranges from 1 mM to 10 mM. The same tendency is also observed when ethanol is used as the solvent, and the liposome size hardly depends on the 10331

dx.doi.org/10.1021/ie401876z | Ind. Eng. Chem. Res. 2013, 52, 10329−10332

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(6) Kremer, J.; Esker, M.; Pathmamanoharan, C.; Wiersema, P. Vesicles of variable diameter prepared by a modified injection method. Biochemisty 1977, 16, 3932. (7) Szoka, F., Jr.; Papahadjopoulos, D. Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc. Natl. Acad. Sci. USA. 1978, 75, 4194. (8) Frederiksen, L.; Anton, K.; van. Hoogevest, P.; Keller, H. R.; Leuenberger, H. Preparation of liposomes encapsulating water-soluble compounds using supercritical carbon dioxide. J. Pharm. Sci. 1997, 86, 921. (9) Otake, K.; Imura, T.; Sakai, H.; Abe, M. Development of a new preparation method of liposomes using supercritical carbon dioxide. Langmuir 2001, 17, 3898. (10) Funakoshi, K.; Suzuki, H.; Takeuchi, S. Formation of giant lipid vesiclelike compartments from a planer lipid membrane by a pulsed jet flow. J. Am. Chem. Soc. 2007, 129, 12608. (11) Stachowiak, J. C.; Richmond, D. L.; Li, T. H.; Liu, A. P.; Parekh, S. H.; Fletcher, D. A. Unilamellar vesicles formation and encapsulation by microfluidic jetting. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 4697. (12) Shum, H. C.; Lee, D.; Yoon, I.; Kodger, T.; Weitz, D. A. Double emulsion template monodipserse phospholipid vesicles. Langmuir 2008, 24, 7651. (13) Seth, A.; Béalle, G.; Santacach-Carreras, E.; Abou-Hassan, A.; Ménager, C. Desing of vesicles using capillary microfluidic devices: From magnetic to multifunctional vesicles. Adv. Mater. 2012, 24, 3544. (14) Jahn, A.; Vreeland, W. N.; Gaitan, M.; Locascio, L. E. Controlled vesicle self-assembly in microfluidic channels with hydrodynamic focusing. J. Am. Chem. Soc. 2004, 126, 2674. (15) Nakashima, T.; Shimizu, M.; Kukizaki, M. Membrane emulsification by microporous glass. Key Eng. Mater. 1991, 61/62, 513. (16) Nakashima, T.; Shimizu, M.; Kukizaki, M. Particle control of emulsion by membrane emulsification and its applications. Adv. Drug Delivery Rev. 2000, 45, 47. (17) Kakazu, E.; Murakami, T.; Akamatsu, K.; Sugawara, T.; Kikuchi, R.; Nakao, S. Preparation of silver nanoparticles using the SPG membrane emulsification technique. J. Membr. Sci. 2010, 354, 1. (18) Akamatsu, K.; Kaneko, D.; Sugawara, T.; Kikuchi, R.; Nakao, S. Three preparation methods for monodispersed chitosan microspheres using the Shirasu Porous Glass membrane emulsification technique and mechanisms of microsphere formation. Ind. Eng. Chem. Res. 2010, 49, 3236. (19) Akamatsu, K.; Chen, W.; Suzuki, Y.; Ito, T.; Nakao, A.; Sugawara, T.; Kikuchi, R.; Nakao, S. Preparation of monodisperse chitosan microcapsules with hollow structures using the SPG membrane emulsification technique. Langmuir 2010, 26, 14854. (20) Akamatsu, K.; Maruyama, K.; Chen, W.; Nakao, A.; Nakao, S. Drastic difference in porous structure of calcium alginate microspheres prepared with fresh or hydrolyzed sodium alginate. J. Colloid Interface Sci. 2011, 363, 707. (21) Akamatsu, K.; Ikeuchi, Y.; Nakao, A.; Nakao, S. Size-controlled and monodisperse enzyme-encapsulated chitosan microspheres developed by the SPG membrane emulsification technique. J. Colloid Interface Sci. 2012, 371, 46. (22) Chang, E. P.; Hatton, T. A. Membrane emulsification and solvent pervaporation processes for the continuous synthesis of functional magnetic and Janus nanobeads. Langmuir 2012, 28, 9748. (23) Cheng, C.-J.; Chu, L.-Y.; Ren, P.-W.; Zhang, J.; Hu, L. Preparation of monodisperse thermo-sensitive poly(N-isopropylacrylamide) hollow microcapsules. J. Colloid Interface Sci. 2007, 313, 382. (24) Yamamoto, T.; Ohmori, T.; Kim, Y. H. Preparation and characterization of monodisperse carbon cryogel microspheres. Microporous Mesoporous Mater. 2008, 112, 211. (25) Hwang, T.; Park, T.-J.; Koh, W.-G.; Cheong, I. W.; Choi, S.-W.; Kim, J. H. Fabrication of nano-scale liposomes containing doxorubicin using Shirasu porous glass membrane. Colloids Surf., A 2011, 392, 250. (26) Cheng, C. J.; Chu, L. Y.; Zhang, J.; Zhou, M. Y.; Xie, R. Preparation of monodisperse poly(N-isopropylacrylamide) microspheres and microcapsules via Shirasu-porous-glass membrane emulsification. Desalination 2008, 234, 184.

structure, e.g., the diameter and lamellarity, as shown in Figures 2−4. There is little difference between IPA and ethanol in terms of solubility in water and dielectric constant. One possible reason for the observed results is that small amounts of the alcohols remain in the lipid membranes and that they affect the membrane fluidity. However, the detailed mechanism of how the alcohol used as the lipid solvent affects the membrane fluidity is unclear at this stage, and further studies need to be carried out.

4. CONCLUSION We succeeded in developing a facile and novel preparation method for monodispersed liposomes by permeation of a lipid−alcohol solution through an SPG membrane. This method is template free, and only uses the mixing of lipid− alcohol solutions and water at the interface formed near the SPG membrane surface, inducing spontaneous liposome formation. When we use IPA and ethanol as the solvents for the lipids, this facile method, regardless of the differences between the solvents, provides monodispersed multilamellar vesicles with diameters of around 120 nm; these are much smaller than the pore sizes of the SPG membranes. This is probably because of the similar solubilities of the alcohols in water. However, the effects of the solvents on the diameterchange behaviors of the liposomes in freeze−thaw cycling is clearly observed. This indicates that the alcohol used for this method would affect the membrane fluidity of the resultant liposomes.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +81-42-628-4584. Fax: +81-42-628-4542. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Prof. H. Umakoshi (Osaka University) for his helpful discussions. Part of this research was supported by a Grant-in-Aid for Challenging Exploratory Research (No. 23656496) from the Japan Society for the Promotion of Science (JSPS).

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REFERENCES

(1) Umakoshi, H.; Morimoto, K.; Yasuda, N.; Shimanouchi, T.; Kuboi, R. Development of liposome-based mimics of superoxide dismutase and peroxidase based on the “LIPOzyme” concept. J. Biotechnol. 2010, 147, 59. (2) Umakoshi, H.; Morimoto, K.; Ohama, Y.; Nagami, H.; Shimanouchi, T.; Kuboi, R. Liposome modified with Mn−porphyrin complex can simultaneously induce antioxidative enzymes-like activity of both superoxide dismutase and peroxidase. Langmuir 2008, 24, 4451. (3) Sugaya, H.; Umakoshi, H.; Tohtake, Y.; Shimanouchi, T.; Kuboi, R. Characterization of hollow fiber immobilized liposome membrane by using aqueous two-phase partitioning sytem. Solv. Extr. Res. Dev. Jpn 2009, 16, 103. (4) Bangham, A. D.; Standish, M. M.; Watkins, J. C. Diffusion of univalent ions across the lamellae of swollen phospholipids. J. Mol. Biol. 1965, 13, 238. (5) Batzri, S.; Korn, E. Single bilayer liposomes prepared without sonication. Biochim. Biophys. Acta 1973, 298, 1015. 10332

dx.doi.org/10.1021/ie401876z | Ind. Eng. Chem. Res. 2013, 52, 10329−10332