Synthesis of Liposome Reinforced with Cholesterol and Application to Transmission Electron Microscopy Observation
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Synthesis of Liposome Reinforced with Cholesterol and Application to Transmission Electron Microscopy Observation Marina Kamogawa1, Takuji Ube1, Junichi Shimanuki2, Takashi Harumoto1, Makoto Yuasa3, and Takashi Ishiguro1 1 Department of Materials Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan 2 Material Analysis Department, NISSAN ARC, LTD., 1 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan 3 Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan ABSTRACT Liposome was synthesized by using mixture of dipalmitoylphosphatidylcholine and cholesterol in the ultrapure water or physiological saline. Phase transformation temperature and vibrational mode of dipalmitoylphosphatidylcholine molecule were detected by using transmission Fourier-transform infrared spectroscopy for aqueous solution, which we developed. The liposomes were fixed on an amorphous carbon mesh for ultra-high resolution transmission electron microscopy observation and stained with platinum thymidine blue. As-prepared liposomes reinforced with cholesterol were spherical in shape with size larger than 100 nm in diameter and still stable in the vacuum. Under the strong electron irradiation condition, the solution enclosed in the liposomes became unstable and then collapsed. On the other hand, the liposome synthesized in the physiological saline sometimes contains crystallized salt. As a result, the liposome shows proper strength to hold wet material in itself in a vacuum and can be used for the transmission electron microscopy observation. INTRODUCTION It is well known that liposome is composed of phospholipid bilayer, which is the basic structure of the cell-wall [1, 2], and is applied to the micro-capsule in the drug delivery system (DDS) [3-6] and in cosmetics [7, 8]. Since the function of biological soft matter usually depends on its three-dimensional conformation, nanoscale observation of biological soft matter such as protein and DNA has been studied intensively [9, 10]. Especially, transmission electron microscopy (TEM) offers images with sub-nanometer resolution, it is one of excellent methods for high resolution observation. There is, however, inevitable problem, i.e., the specimen should be settled in a vacuum. This means that the observation of raw biological soft matter is difficult. Therefore, the direct nanoscale observation method for biological soft matter is important technique which should be developed and, in this study, we try to use the liposome as a microcapsule for TEM observation of wet material. EXPERIMENT First of all, conventional liposome was synthesized by using 2, 3-di(hexadecanoyloxy)propyl 2-(trimethylazaniumyl)ethyl phosphate, i.e., dipalmitoylphosphatidylcholine (DPPC). However
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these liposomes have weak resistance to electron radiation. In this study, we examined reinforcement of the liposome with cholesterol. However phase diagr
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