Hydroethidine, a reduced form of ethidium bromide, was used as a vital dye in fluorescence assays that allowed visual and semiquantitative monitoring of dye uptake and accumulation by fluorescence microscopy, flow cytometry, image analysis, and microfluorimetry. The excitation and emission filters were chosen to detect hydroethidine and exclude ethidium. Microscopically, there were differences in fluorescence intensities and fluorescence patterns among various tumor cell lines. The fluorescence pattern varied from homogeneous blue in the cytoplasm to blue plus brilliant packets of bluish-white distributed in the cytoplasm. Nuclear staining varied from brown to reddish orange fluorescence. These differences were confirmed by flow cytometry and image analysis. A preliminary survey of various tumors indicated that uptake and accumulation of hydroethidine were dependent on concentration of the dye, duration of cell exposure to the dye, and metabolic state of the cells. Microfluorimetry made possible monitoring of 96 samples in a microculture plate in 30 seconds; thus, this method allows large numbers of samples to be read, with a tremendous savings in time and reagents. The results obtained from the different techniques used were corroborative; therefore, any one of the above techniques may be used in an assay.
The ability of membrane lipids to adopt nonbilayer configurations suggests dynamic roles for lipids in many functional abilities of biological membranes. In this work evidence supporting the involvement of lipids in three types of membrane transport process is presented and discussed. These transport processes include facilitated transbilayer transport of polar molecules, transport mechanisms involving fusion events, and transport possibilities arising from alternative membrane morphology. In particular it is shown that lipids such as cardiolipin, which adopt the hexagonal HII phase in the presence of Ca2+, may be logically proposed to facilitate Ca2+ transport across membranes via an inverted micellar intermediate. Alternatively, in transport processes such as exocytosis the ability of Ca2+ to generate membrane instabilities favouring nonbilayer alternatives suggests a crucial role of phospholipid in the fusion event vital to exocytotic release. Finally, nonbilayer lipid structures may be suggested to favour formation of isolated compartments connected by a continuous membrane where lateral diffusion processes can lead to transport. These various possibilities are summarized in a "metamorphic mosaic" model of biological membranes.
The influence of vesicle lipid composition, size and drug-to-lipid ratio on the antitumour activity of liposomal vincristine was assessed in the murine L1210 ascitic leukemia model. A pH gradient-dependent entrapment procedure was used to encapsulate vincristine and allowed such vesicle properties to be independently varied. Free vincristine delivered i.v. at the maximum tolerated dose (2.0 mg/kg) resulted in a 27.8% increase in the life span (ILS) of mice inoculated i.p. with L1210 cells. Encapsulation of the drug in egg phosphatidylcholine/cholesterol vesicles did not significantly increase the antitumour efficacy of vincristine (ILS, 38.9%). In contrast, administration of vincristine entrapped in vesicles composed of distearoylphosphatidylcholine (DSPC)/cholesterol resulted in ILS values as high as 133%. This enhanced antitumour activity of the DSPC/cholesterol formulations was sensitive to the size of the liposomes; increasing the vesicle size from 100 nm to 1 micron decreased the ILS from 133.3% to 55.6% at a drug dose of 2.0 mg/kg. Decreasing the drug-to-lipid ratio from 0.1:1 to 0.05:1 (w/w) had negligible effects on the activity of liposomal vincristine; however, a further decrease in the drug-to-lipid ratio to 0.01:1 (w/w) decreased the antitumour potency at all drug doses studied. Pharmacology studies indicated that the antitumour activities of free and various liposomal forms of vincristine correlated well with the residence time of the drug in the circulation. These studies indicate that efforts to enhance the therapeutic activity of vincristine through liposome encapsulation must address not only the circulation lifetime of the vesicle systems but also the capacity of the liposomes to retain entrapped drug in vivo.
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