INTRACELLULAR DELIVERY AND ANTIBACTERIAL ACTIVITY OF GENTAMICIN ENCAPSULATED IN PH-SENSITIVE LIPOSOMES

Cell membranes are relatively impermeable to the antibiotic gentamicin , a factor that, along with the toxicity of gentamicin, precludes its use against many important intracellular bacterial infections. Liposom al encapsulation of this drug was used in order to achieve intracellul ar antibiotic delivery and therefore increase the drug's therapeutic a ctivity against intracellular pathogens. Gentamicin encapsulation in s everal dipalmitoylphosphatidylcholine (DPPC) and pH-sensitive dioleoyl phosphatidylethanolamine (DOPE)-based carrier systems was characterize d, To systematically test the antibacterial efficacies of these formul ations, a tissue culture assay system was developed wherein murine mac rophage-like J774A.1 cells were infected with bacteria and were then t reated with encapsulated drug. Of these formulations, DOPE-N-succinyl- DOPE and DOPE-N-glutaryl-DOPE (70:30;mol:mol) containing small amounts of polyethyleneglycol-ceramide showed appreciable antibacterial activ ities, killing greater than 75% of intracellular vacuole-resident wild -type Salmonella typhimurium compared to the level of killing of the c ontrol formulations. These formulations also efficiently eliminated in tracellular infections caused by a recombinant hemolysin-expressing S, typhimurium strain and a Listeria monocytogenes strain, both of which escape the vacuole and reside ire the cytoplasm, Control non-pH-sensi tive liposomal formulations of gentamicin had poor antibacterial activ ities. A fluorescence resonance energy transfer assay indicated that t he efficacious formulations undergo a pH-dependent lipid mixing and fu sion event, Intracellular delivery of the fluorescent molecules encaps ulated in these formulations was confirmed by confocal fluorescence mi croscopy and was shown to be dependent on endosomal acidification. Thi s work shows that encapsulation of membrane-impermeative antibiotics i n appropriately designed lipid based delivery systems can enable their use in treating intracellular infections and details the development of a general assay for testing the intracellular delivery of encapsula ted drug formulations.