Resonance energy-transfer and fluorescence intensity studies of the transport of liposome-encapsulated molecules into isolated mouse liver nuclei.
We present evidence that liposomes (composed of egg yolk L-alpha-phosphatidylcholine/phosphatidylethanolamine/cholesterol, in a molar ratio of 4:5:1) fuse with isolated mouse liver nuclei at low pH. Using the resonance energy-transfer assay, we determined the rate and extent of liposome and nuclear membrane lipid mixing. Fusion was substantial when the pH was below 5. The half-time of lipid mixing decreased by acidification of the solvent, reaching about 2 min at pH 4.5. In order to study the transport of the liposome-aqueous contents to the interior of the nuclei during the process, we developed fluorescence assays in which fluorescein isothiocyanate labeled dextrans of 150 kDa molecular mass (FITC-D150) were encapsulated in liposomes. These liposomes also included in their bilayers the fluorescent lipid N-tetramethylrhodamine-L-alpha-dipalmitoylphosphatidylethanolamine (N-Rh-DPPE). After incubation of these liposomes with mouse liver nuclei (pH 4.5, 37 degrees C, 30 min), we measured the fluorescence spectra of a suspension of washed nuclei and of nuclei treated by the detergent Triton X-100 (membrane-denuded nuclei). These Triton X-100 treated nuclei had no N-Rh-DPPE fluorescence while they showed a FITC-D150 fluorescence which amounted to 20% of that of the intact nuclei. In another assay, a laser beam was focused on single nuclei by a microscope epiexcitation device. The variation of the N-Rh-DPPE and FITC-D150 fluorescence with the nuclear radius was determined with the microphotometric attachment of the microscope.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism*; D003911:Dextrans; D004735:Energy Transfer; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008081:Liposomes / Q000008:administration & dosage*; D008099:Liver / Q000378:metabolism*; D051379:Mice; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D013050:Spectrometry, Fluorescence / Q000379:methods
D003911:Dextrans; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008081:Liposomes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; C015219:fluorescein isothiocyanate dextran; D016650:Fluorescein-5-isothiocyanate