SELECTIVE LIPID-PATTERNING FOR HETEROLOGOUS GIANT …
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SELECTIVE LIPID-PATTERNING FOR HETEROLOGOUS GIANT LIPOSOME ARRAY T. Osaki, 1,* K. S. Kuribayashi, 2 R. Kawano, 1 H. Sasaki, 1 K. Kamiya, 1 and S. Takeuchi 1,2 1 Kanagawa Academy of Science and Technology, JAPAN, and 2 Institute of Industrial Science, The University of Tokyo, JAPAN ABSTRACT This paper describes a methodology that realizes an array of heterologous giant liposomes with gentle hydration. The system consists of an array of micropores on a thin polymer film, which is overlaid on a pair of comb ITO-electrodes. An electrospray deposition (ESD) method allows selective lipid patterning of green and red fluorescent-labeled lipids at the mi- cropores that expose either of the target ITOs. With hydration of the lipid film, we successfully obtained an alternately- colored giant liposome array on top of the pattern. KEYWORDS: Selective patterning, Electrospray, Giant liposome, Heterologous array INTRODUCTION Giant Liposomes Giant liposome or giant lipid vesicle is one of the self- assembled structures of lipid molecules, encapsulating an aqueous solution by a single or multiple lipid bilayer mem- branes with a diameter ranging between a few micrometers and a few hundred micrometers [1]. Such microcontainers find a number of applications in the fields of drug delivery, cosmetics and food products as well as the biological fun- damental studies [2]. Selective Patterning for Giant Liposome Formation Gentle hydration and electroformation are the most common methods for the giant liposome preparation. The gentle hydration simply takes advantage of a hydration process of a dried lipid film with an aqueous solution while the electroformation applies a low-frequency AC voltage during the hydration [1]. Although these methods have been widely used, they both have difficulty in controlling the shape and/or the size distribution of the formed liposomes [1, 3, 4]. Patterning of lipids prior to the hydration process will be one of the solutions of those problems. There are several techniques reported to obtain such lipid patterns, for instance, hydrophilic/hydrophobic patterning of a substrates’ surface [4], or a lift-off method with a patterned polymer film [5], or an application of a microcontact printing [6]. The results indicated that the selective patterning would improve the uniformi- ty of the formed liposomes, yet neither methods have successfully regulated the lipid patterning/deposition process. In the previous work, we therefore developed an alternative method that enables the precise control of the patterning process by the integration of an electrospray deposition (ESD) technique and a microfabrication process [7]; by the ESD, the spray of lipids was electrically led to only the bottom of microwells which was a conductive ITO-glass slide (details in the Experimental section). With a simple hydration process of these dried patterns, we succeeded in formation of giant lipo- somes on top of the micropores with a narrow range of the size distribution and easily obtained the desired sizes of liposomes by changing the microwell diameter (between 5 and 30 m in diameter). In this paper, we set the next goal to present a further application of the ESD method combined with a microfabrication technique. We focus on the positional selectivity of the pattern by the method, which is another benefit of using ESD, aim- ing for a heterologous giant liposome array with designing the target ITO electrodes (Fig. 1). EXPERIMENTAL Fig. 2 shows the fabrication flow. The process consists of two steps: 1) Fabrication of the patterned substrate by using common photolithography and wet-etching, and 2) a lipid deposition process with the ESD technique. 978-0-9798064-4-5/μTAS 2011/$20©11CBMS-0001 1137 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 2-6, 2011, Seattle, Washington, USA
Transcript of SELECTIVE LIPID-PATTERNING FOR HETEROLOGOUS GIANT …
SELECTIVE LIPID-PATTERNING FOR HETEROLOGOUS GIANT LIPOSOME ARRAY
T. Osaki,1,* K. S. Kuribayashi,2 R. Kawano,1 H. Sasaki,1 K. Kamiya,1 and S. Takeuchi1,2 1 Kanagawa Academy of Science and Technology, JAPAN, and
2 Institute of Industrial Science, The University of Tokyo, JAPAN ABSTRACT
This paper describes a methodology that realizes an array of heterologous giant liposomes with gentle hydration. The system consists of an array of micropores on a thin polymer film, which is overlaid on a pair of comb ITO-electrodes. An electrospray deposition (ESD) method allows selective lipid patterning of green and red fluorescent-labeled lipids at the mi-cropores that expose either of the target ITOs. With hydration of the lipid film, we successfully obtained an alternately-colored giant liposome array on top of the pattern. KEYWORDS: Selective patterning, Electrospray, Giant liposome, Heterologous array
INTRODUCTION Giant Liposomes
Giant liposome or giant lipid vesicle is one of the self-assembled structures of lipid molecules, encapsulating an aqueous solution by a single or multiple lipid bilayer mem-branes with a diameter ranging between a few micrometers and a few hundred micrometers [1]. Such microcontainers find a number of applications in the fields of drug delivery, cosmetics and food products as well as the biological fun-damental studies [2].
Selective Patterning for Giant Liposome Formation
Gentle hydration and electroformation are the most common methods for the giant liposome preparation. The gentle hydration simply takes advantage of a hydration process of a dried lipid film with an aqueous solution while the electroformation applies a low-frequency AC voltage during the hydration [1]. Although these methods have been widely used, they both have difficulty in controlling the shape and/or the size distribution of the formed liposomes [1, 3, 4]. Patterning of lipids prior to the hydration process will be one of the solutions of those problems. There are several techniques reported to obtain such lipid patterns, for instance, hydrophilic/hydrophobic patterning of a substrates’ surface [4], or a lift-off method with a patterned polymer film [5], or an application of a microcontact printing [6]. The results indicated that the selective patterning would improve the uniformi-ty of the formed liposomes, yet neither methods have successfully regulated the lipid patterning/deposition process.
In the previous work, we therefore developed an alternative method that enables the precise control of the patterning process by the integration of an electrospray deposition (ESD) technique and a microfabrication process [7]; by the ESD, the spray of lipids was electrically led to only the bottom of microwells which was a conductive ITO-glass slide (details in the Experimental section). With a simple hydration process of these dried patterns, we succeeded in formation of giant lipo-somes on top of the micropores with a narrow range of the size distribution and easily obtained the desired sizes of liposomes by changing the microwell diameter (between 5 and 30 �m in diameter).
In this paper, we set the next goal to present a further application of the ESD method combined with a microfabrication technique. We focus on the positional selectivity of the pattern by the method, which is another benefit of using ESD, aim-ing for a heterologous giant liposome array with designing the target ITO electrodes (Fig. 1).
EXPERIMENTAL
Fig. 2 shows the fabrication flow. The process consists of two steps: 1) Fabrication of the patterned substrate by using common photolithography and wet-etching, and 2) a lipid deposition process with the ESD technique.
978-0-9798064-4-5/µTAS 2011/$20©11CBMS-0001 1137 15th International Conference onMiniaturized Systems for Chemistry and Life Sciences
October 2-6, 2011, Seattle, Washington, USA
First, a pair of comb electrodes was patterned on an ITO-glass slide with a wet-etching process. Then, a poly(chloro-p-xylylene) film was coated on the slide by a chemical vapor deposition method. By a standard UV-lithography process, micropores were fabricated on the polymer film (thick. 1 �m, diam. 20 �m), where those pores were overlaid on the elec-trodes (see the images in Fig. 2c).
The ESD setup is shown in Fig. 3. The technique ap-plies a high DC voltage between a lipid solution filled in a thin capillary and the target electrodes, enabling a selective deposition of lipids at the conductive surfaces. The spray is precisely regulated by the applied electric field, the inner di-ameter of the capillary tip, and the properties of the sprayed solution, and the sprayed mist accumulates at the conductive surface in a dry state and directly forms the pattern. In this work, ESD was performed with phospholipids in a chloro-form/acetonitrile (95/5) mixture (0.5 mg/mL DOPC, 2 kV/cm applied, 15 s of spray), where 1% of either lissamine-rhodamine B (red) or nitrobenzoxadiazol (green) labeled lipid was mixed for fluorescent imaging.
Hydration of dried lipids for the liposome formation was simply performed by infusion of MilliQ water to the wells that were punched in a silicone-rubber sheet placed on the lipid-patterns. Images of the formed liposomes were taken through the ITO-glass slide with a confocal inverted microscope (SP5, Leica Microsystems, Germany).
RESULTS AND DISCUSSION
As shown in Fig. 4a, the depositions of the different colors of lipids were switched by the application of a slight negative vol-tage (-10 V/cm) to the target ITO electrode while the other one was grounded. We observed the dried lipid films with a micro-scope after each ESD process (Fig. 4b top and bottom). The images clearly showed that the desired lipid solutions were selec-tively deposited at the objective patterns by switching the target electrode. We found that the negative voltage applied to the target ITO was important to suppress the cross-contamination at the pair ITO during the ESD. Note that the lipid solutions were simply sprayed without any kinds of masking.
Liposome formation was then performed in MilliQ water. Fig. 5a shows a birds-eye view image of the giant liposome array, reconstructed from a series of confocal microscopic images. As clearly seen, alternately-colored liposomes were arrayed on the micropores. Described in the previous work, the developed ESD method easily realizes an array of uniform-size giant liposomes,
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whose diameter is close to the micropore size, by adjusting the lipid deposition conditions mentioned above [7]. The red and the green fluorescent-labeled liposomes were also formed with a well-controlled size in this work (Fig. 5b).
CONCLUSION
We succeeded in the formation of a heterologous giant liposome array under a gentle hydration. The result demonstrated the potential of the selective lipid patterning method that takes advantage of the ESD technique in combination with a com-mon microfabrication process. Since the formed liposomes are statically arrayed on a substrate, the system will be suitable for time-resolved and/or statistical analyses. We consider that the developed system would find various applications from fundamental liposome studies to functional analyses of membrane proteins. ACKNOWLEDGEMENTS
The authors acknowledge the technical support provided by Ms. U. Nose, and Ms. Y. Komaki, Ms. M. Uchida, and Ms. W. Mukai. This work was supported by JSPS (Grant-in-Aid for Young Scientists B; 23710154), Japan. REFERENCES [1] P. L. Luisi, P. Walde, Giant Vesicles, John Willy and Sons Inc., New York, 2000; N. Duzgunes, Methods in Enzymolo-
gy Volume 367 Liposomes Part A, Academic Press, California, 2003. [2] A. Karlsson, R. Karlsson, M. Karlsson, A-S. Cans, A. Strömberg, F. Ryttsén, O. Orwar, “Networks of Nanotubes and
Containers”, Nature 2001, 409, 150- 152.; I. A. Chen, K. Salehi-Ashtiani, J. W. Szostak, “RNA Catalysis in Model Protocell Vesicles”, J. Am. Chem. Soc. 2005, 127, 13213-13219.; G. Tresset, S. Takeuchi, “Utilization of Cell-sized Li-pid Containers for Nanostructure and Macromolecule Handling in Microfabricated Devices”, Anal. Chem. 2005, 77, 2795- 2801.
[3] K. Kuribayashi, G. Tresset, H. Fujita, S. Takeuchi, “Electroformation of Giant Liposomes in Microfluidic Channels”, Meas. Sci. Technol. 2006, 17, 3121-3126
[4] M. Le Berre, A. Yamada, L. Rech, Y. Chen, D. Baigl, “Electroformation of Giant Phospholipid Vesicles on a Silicon Substrate: Advantages of Controllable Surface Properties”, Langmuir 2008, 24, 2643-2649.
[5] K. Kuribayashi, S. Takeuchi, “Electroformation of Solvent-Free Lipid Membranes over Microaperture Array”, Proc. IEEE MEMS 2008, Tucson, 296-299.
[6] P. Taylor, C. Xu, P. D. I. Fletcher, V. Paunov, “Fabrication of 2D Arrays of Giant Liposomes on Solid Substrates by Microcontact Printing”, Phys. Chem. Chem. Phys. 2003, 5, 4918-4922.
[7] T. Osaki, K. S. Kuribayashi, R. Kawano, H. Sasaki, S. Takeuchi, “Uniformly-Sized Giant Liposome Formation with Gentle Hydration”, Proc. IEEE MEMS 2011, Cancun, 103-106.
CONTACT *T Osaki, Bio Microsystems Project, Kanagawa Academy of Science and Technology (KAST), 3-2-1 Sakado, Takatsu, Ka-wasaki 213-0012, Japan, Tel: +81-44-819-2037, Email: [email protected]
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