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We developed a model to test whether non-membrane-permeative therapeutic agents such as gentamicin could be delivered into mammalian cells by means of bacterial membrane vesicles. Many gram-negative bacteria bleb off membrane vesicles (MVs) during normal growth, and the quantity of these vesicles can be increased by brief exposure to gentamicin (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 1995), which can be entrapped within the MVs. Gentamicin-induced MVs (g-MVs) were isolated from Shigella flexneri and contained 85 +/- 2 ng of gentamicin per microgram of MV protein. Immunogold electron microscopic labeling of thin sections with antibodies specific to S. flexneri lipopolysaccharide (LPS) demonstrated the adherence and subsequent engulfment of MVs by the human Henle 407 intestinal epithelial cell line. Further incubation of g-MVs with S. flexneri-infected Henle cells revealed that the g-MVs penetrated throughout the infected cells and reduced the intracellular pathogen by approximately 1.5 log10 CFU in the first hour of incubation. Antibiotic was detected in the cytoplasms of host cells, indicating the intracellular placement of the drug following the penetration of g-MVs. Soluble antibiotic, added as a fluid to the tissue culture growth medium, had no effect on intracellular bacterial growth, confirming the impermeability of the cell membranes of the tissue to gentamicin. Western blot analysis of MVs with S. flexneri Ipa-specific antibodies demonstrated that the invasion protein antigens IpaB, IpaC, and IpaD were present in MVs. Being bilayered, with outer faces composed of LPS and Ipa proteins, these MVs were readily engulfed by the otherwise impermeable membranes and eventually liberated their contents into the cytoplasmic substance of the host tissue.

Delivery of the Non-Membrane-Permeative Antibiotic Gentamicin into Mammalian Cells by Using Shigella flexneri Membrane Vesicles