TY - JOUR
T1 - Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope
AU - Akashi, Ken Ichirou
AU - Miyata, Hidetake
AU - Itoh, Hiroyasu
AU - Kinosita, Kazuhiko
N1 - Funding Information:
We thank Dr. Y. Kirino (University of Tokyo) and Dr. S. Ishiwata (Waseda University) for discussions, Dr. A. Ikegami (Keio University School of Medicine) and Dr. Y. Inoue (The Institute of Physical and Chemical Research) for support, Mr. M. Hosoda and Mr. K. Atusumi (Hamamatsu Photonics K. K.) for help in developing the image analysis system, and Mr. J. Noguchi (Keio University) for help in the initial stage of this research. This research was supported by a special Grant-in-Aid for Innovative Collaborative Research Projects from Keio University, a grant from Ka- nagawa Academy of Science and Technology, a grant from Terumo life ScienceFoundation,grants-in-aidfromtheMinistryofEducation, Science and Culture of Japan, and special coordination funds for promoting science andtechnologyfromtheAgencyofScienceandTechnologyof Japan.
PY - 1996/12
Y1 - 1996/12
N2 - Unilamellar liposomes with diameters of 25-100 μm were prepared in various physiological salt solutions, e.g., 100 mM KCl plus 1 mM CaCl2. Successful preparation of the giant liposomes at high ionic strengths required the inclusion of 10-20% of a charged lipid, such as phosphatidylglycerol, phosphatidylserine, phosphatidic acid, or cardiolipin, in phosphatidylcholine or phosphatidylethanolamine. Three criteria were employed to identify unilamellar liposomes, yielding consistent results. Under a phase-contrast microscope those liposomes that showed the thinnest contour and had a vigorously undulating membrane were judged unilamellar. When liposomes were stained with the lipophilic fluorescent dye octadecyl rhodamine B, fluorescence intensities of the membrane of individual liposomes were integer multiples (up to four) of the lowest ones, the least fluorescent liposomes being those also judged unilamellar in the phase-contrast image. Micropipette aspiration test showed that the liposomes judged unilamellar in phase and fluorescence images had an area elastic modulus of ~160 dyn/cm, in agreement with literature values. The giant liposomes were stable and retained a concentration gradient of K+ across the membrane, as evidenced in fluorescence images of the K+-indicator PBFI encapsulated in the liposomes. Ionophore-induced K+ transport and associated volume change were observed in individual liposomes.
AB - Unilamellar liposomes with diameters of 25-100 μm were prepared in various physiological salt solutions, e.g., 100 mM KCl plus 1 mM CaCl2. Successful preparation of the giant liposomes at high ionic strengths required the inclusion of 10-20% of a charged lipid, such as phosphatidylglycerol, phosphatidylserine, phosphatidic acid, or cardiolipin, in phosphatidylcholine or phosphatidylethanolamine. Three criteria were employed to identify unilamellar liposomes, yielding consistent results. Under a phase-contrast microscope those liposomes that showed the thinnest contour and had a vigorously undulating membrane were judged unilamellar. When liposomes were stained with the lipophilic fluorescent dye octadecyl rhodamine B, fluorescence intensities of the membrane of individual liposomes were integer multiples (up to four) of the lowest ones, the least fluorescent liposomes being those also judged unilamellar in the phase-contrast image. Micropipette aspiration test showed that the liposomes judged unilamellar in phase and fluorescence images had an area elastic modulus of ~160 dyn/cm, in agreement with literature values. The giant liposomes were stable and retained a concentration gradient of K+ across the membrane, as evidenced in fluorescence images of the K+-indicator PBFI encapsulated in the liposomes. Ionophore-induced K+ transport and associated volume change were observed in individual liposomes.
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U2 - 10.1016/S0006-3495(96)79517-6
DO - 10.1016/S0006-3495(96)79517-6
M3 - Article
C2 - 8968594
AN - SCOPUS:0029752766
VL - 71
SP - 3242
EP - 3250
JO - Biophysical Journal
JF - Biophysical Journal
SN - 0006-3495
IS - 6
ER -