Resonance energy-transfer and fluorescence intensity studies of the transport of liposome-encapsulated molecules into isolated mouse liver nuclei

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Abstract
We present evidence that liposome (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 fluoresecent lipid N-tetramethylrhodamine-L-.alpha.-dipalmitoylphosphatidylethanolamine (N-Rh-DPPE). After incubation of these liposomes with mouse liver nuclei (pH 4.5, 37.degree. 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 X100 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. For the N-Rh-DPPE which was confined on the nuclear surface, the fluorescence intensity decrased strongly when the nucleus radius increased, as expected from the optical sectioning property of the microscope objective. This decrease was much weaker with the FITC-D150 fluorescence, showing that the dextran was partly distributed in the nuclear volume. By fitting these variations with formulas giving the light collection efficiency of the objective [Koppel, D. E., Axelrod, D., Schlessinger, J., Elson, E. L., and Webb, W. W. (1976) Biophys. J. 16, 1315-1329], we determined that 30% of the nucleus-associated dextrans were in the nucleus interior. To our knowledge, these are the first reported results showing that liposomes fuse with isolated mouse liver nuclei and that the liposome contents can be transferred into the nucleus interior. Our work suggests that liposome fusion with the nuclear membrane might be one of the steps in the process of transfer of liposome-encapsulated genes to eukaryote cells [Wong, T. K., Nicolau, C., and Hofschnieder, P. H. (1980) Gene 10, 87-94; Soriano, P., Dijkstra, J., Legrand, A., Spanjer, H., Lodos-Gagliardi, D., Roerdink, F., Scherphof, G., and Nicolau, C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7128-7131].