Cationic Vesicles as Bactericides L. M. S. Martins, † E. M. Mamizuka, ‡ and A. M. Carmona-Ribeiro* ,† Departamento de Bioquı ´mica, Instituto de Quı ´mica, Universidade de Sa ˜ o Paulo, CP 26077, CEP 05599-970 Sa ˜ o Paulo SP, Brazil, and Departamento de Ana ´ lises Clı ´nicas e Toxicolo ´ gicas, Faculdade de Cie ˆ ncias Farmace ˆ uticas, USP, CP 66083, Sa ˜ o Paulo SP, Brazil Received April 7, 1997. In Final Form: August 8, 1997 X Dioctadecyldimethylammonium bromide (DODAB), a liposome-forming synthetic amphiphile, kills Escherichia coli, Salmonella thyphimurium, Pseudomonas aeruginosa, and Staphylococcus aureus in the micromolar range of DODAB concentrations. For the four species at cell concentrations higher than 10 7 bacteria/mL in the interaction mixtures, 5 μM DODAB, and 5 h of interaction time between bacteria and vesicles, 0% survival (no counts for viable cells) was obtained. The mechanism of cell death does not involve cell lysis or vesicle rupture as evaluated from measurements of cell leakage of phosphorylated compounds and from a vesicle disruption assay. The isolated external membrane of E. coli and DODAB cationic small vesicles do interact to yield an increase in the electrophoretic mobility of ghosts as a function of DODAB concentration. Surface charge for the ghosts becomes zero over the micromolar range of DODAB concentrations. Thus vesicle adhesion to the external membrane of the bacteria is certainly the first interaction step. Results on dose and time effects on cell viability generalize the bactericidal effect of cationic DODAB vesicles to four bacteria species of clinical importance. Introduction The quaternary nitrogen moiety is an essential com- ponent for many biologically active compounds, 1 including vitamins, enzymes, phosphatidylcholine, choline, and acetylcholine. Medicinal chemists using the principle of structure-activity relationships have synthesized many quaternary ammonium compounds that will mimic certain biological effects. 2 In 1935, the antibacterial activity of the long-chain quaternary ammonium salts was disclosed. 3 The fourth generation of quaternary antimicrobials in- cluded several mono- and dialkyl dimethyl ammonium salts and polymeric quaternary ammonium salts such as the ionenes, which are polyelectrolytes with positively charged nitrogen atoms located in the backbone of the polymeric chain. 4 The antimicrobial, antifungal, and tumoricidal properties of ionenes indicated that the polymers are more active than the corresponding mono- mers, this being interpreted as due to favored adsorption onto the biological surface and the cytoplasmic membrane with subsequent disruption of its integrity. 5 -7 On the other hand, liposomes are relatively well- established as carriers for antimicrobial and anticancer agents. 8,9 They reduce the toxicity of drugs in the target organ by modifying drug distribution and improve the therapeutic index observed with several antimonials, 10,11 imunomodulators, 12,13 antifungal agents, 14,15 and antibiot- ics. 16,17 Liposome encapsulation results in sustained local concentrations of antimicrobial agents. 18-19 After in vivo administration via the intravenous route, conventional liposomes are taken up by the reticuloendothelial system (RES), being potentially useful as antibiotic carriers for treatment of infections involving the RES. 20 Alternatively, vesicle size and phospholipid composition may be con- trolled to change liposome biodistribution and circulation time. 21 A general property of conventional liposomes is that by themselves they are generally inocuous. Recently, physicochemical aspects of the interaction between synthetic cationic vesicles 22 composed of diocta- decyldimethylammonium bromide (DODAB) or chloride (DODAC) and Escherichia coli were described. Adsorption isotherms point to an interaction of high affinity with limiting adsorption values compatible with vesicle adhe- sion to bacteria without vesicle rupture. 23 There are flocculant and bactericidal effects of these vesicles on E. coli that are not related to each other; i.e., cell death is not related to aggregation taking place also for nonag- * To whom correspondence should be addressed. Fax: 055 11 815 5579. E-mail: mcribeir@quim.iq.usp.br. † Departamento de Bioquı ´mica. ‡ Departamento de Ana ´ lises Clı ´nicas e Toxicolo ´gicas. X Abstract published in Advance ACS Abstracts, October 1, 1997. (1) Merianos, J. J. Quaternary ammonium compounds. In Disinfec- tions, Sterilization and Preservation, 4th ed.; Block, S. 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