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The overexpression of lectins by malignant cells was applied for in vitro targeting of liposomes equipped with a saccharide vector and loaded in the lipid phase with a lipid derivative of anticancer agent sarcolysine. The lectin specificity of human leukemia HL-60 and human lung adenocarcinoma ACL cells was revealed by tests with fluorescein-labeled sugar probes. With the help of fluorescent lipid dye it was shown that active saccharide ligands increased the level of the vectored liposome binding to malignant cells by 50-80% as compared to liposomes without vector or with inactive one. The degree of liposome/cell membrane fusion was monitored fluorometrically and was shown to be complete and independent of the vectors. The targeted drug-loaded liposomes had the cytotoxic activity 2-4 times higher as compared to the vector-free ones.

Two new fluorescent lysophosphatidylcholine probes have been synthesized for use as a donor-acceptor pair in fluorescence resonance energy transfer (FRET): 9-anthrylvinyl (LAPC) as donor and 3-perylenoyl (LPPC) as acceptor. The partition coefficients between membrane and aqueous phases were 8.3 x 10(5) and 10.5 x 10(5) for LAPC and LPPC, respectively. The inner leaflets of unilamellar lipid vesicles were labeled with these probes to assess conservation of membrane sidedness after membrane fusion. After medium-sized unilamellar vesicles (MUV) were prepared with a probe in both leaflets, probe in the outer leaflet was removed by repeatedly washing with an excess of unlabeled giant unilamellar vesicles (GUV). MUV and GUV were separated by centrifugation. The probes did not flip-flop across bilayers at 25 degrees C for at least 12 h. MUV containing the ganglioside GT1b were labeled with the LAPC/LPPC pair in the inner leaflet and incubated for 30 min at neutral pH with influenza virus. Fusion was triggered by acidification to pH 5.0 and was monitored by an increase in donor fluorescence in a FRET assay. When the inner leaflets of MUV were labeled by LAPC only, its fluorescence did not change after fusion. However, the fluorescence decreased by 60% when the LAPC was removed from the outer leaflets of the fused membranes by repeated washings with GUV. We conclude that the lipids of the inner and outer leaflets of the fused MUV/virus complexes intermixed.

The inner leaflet of unilamellar lipid vesicles was labeled with fluorescent lysophosphatidylcholines. The probes make a donor-acceptor pair in resonance energy transfer (RET), being labeled with 9-anthrylvinyl (L-APC, donor) and 3-perylenoyl (L-PPC, acceptor) fluorophores. They migrate rapidly between bilayers through the water phase: tau 1/2 of equilibration is approximately 5 min at 37 degrees C. The probe(s) can be removed from the outer leaflet of uniformly labeled medium-size unilamellar vesicles (MUV) by repeated washings with excess unlabeled large unilamellar vesicles (LUV) (separation by centrifugation). The probes flip-flop across bilayers rather slowly. MUV containing the ganglioside GT1b and labeled with the L-APC/L-PPC pair in the inner leaflet were fused with an equal amount of influenza virus; the process was monitored by an increase of the donor fluorescence in RET assay. If inner MUV leaflet was labeled with the anthrylvinyl probe only, the probe fluorescence decreased by half when the probe was removed from the outer leaflets of the fused membranes. This shows that the lipids of the inner and outer leaflets of the MUV randomize in the process of fusion.