Large-scale preparation of asymmetrically labeled fluorescent lipid vesicles.
A method for producing lipid vesicles containing fluorescent phospholipid analogues localized to the inner leaflet of their membrane was developed. Incubation of a 450-fold molar excess of serum albumin with lipid vesicles symmetrically labeled with 1 mol % 1-palmitoyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazolyl)amino-caproyl phosphatidylcholine resulted in the removal of 99% of the fluorescent lipid from the outer leaflet. Asymmetrically labeled vesicles were separated from albumin/lipid complexes by gel filtration chromatography. Vesicles prepared in this manner were unable to transfer fluorescent lipid to cells during liposome-cell incubations. Liposomes asymmetrically labeled with other 4-nitrobenzo-2-oxa-1,3-diazole (NBD)-phospholipid analogues were also prepared. Removal of amino-dodecanoyl-NBD-labeled lipids from the outer leaflet of liposomes required three times more bovine serum albumin, and 48 h of incubation. This method can be used to produce large amounts of asymmetrically labeled liposomes suitable for use in investigating a variety of membrane phenomena.
Dao
H N
HN
Department of Molecular Genetics, Biochemistry, University of Cincinnati College of Medicine, Ohio 45267-0524.
McIntyre
J C
JC
Sleight
R G
RG
D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D006224:Cricetinae; D004735:Energy Transfer; D005456:Fluorescent Dyes; D007700:Kinetics; D008081:Liposomes / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification; D008722:Methods; D008856:Microscopy, Fluorescence; D010069:Oxadiazoles; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000302:isolation & purification
D005456:Fluorescent Dyes; D008081:Liposomes; D010069:Oxadiazoles; C011514:4-nitrobenzofurazan; D012710:Serum Albumin, Bovine