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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 Cieˆ ncias Farmaceˆ uticas, USP, CP 66083, Sa˜ o Paulo SP, Brazil Received April 7, 1997. In Final Form: August 8, 1997X 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 107 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 component 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 included 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 monomers, 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 wellestablished 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 * To whom correspondence should be addressed. Fax: 055 11 815 5579. E-mail:
[email protected]. † 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 Disinfections, Sterilization and Preservation, 4th ed.; Block, S. S., Ed.; Lea & Farbiger: Philadelphia and London, 1991; pp 225-255. (2) Goldstein, A.; Aronow, L.; Kaiman, S. M. Principles of Drugs Action. In Structure Activity Relationships, 2nd ed.; John Wiley & Sons: New York, 1974. (3) Domagk, G. Dtsch. Med. Wonchenschr. 1935, 61, 829-832. (4) Petrocci, A. N.; Clarke, P.; Merianos, J.; Green, H. (1979) Dev. Ind. Microbiol. 1979, 20, 11-14. (5) Ikeda T.; Tazuke, S. Polym. Prepr. 1985, 26, 226-227. (6) Franklin, T. J.; Snow, G. A. Biochemistry of Antimicrobial Action, 4th ed.; Chapman and Hall: London, 1989. (7) Salt, W. G.; Wiseman, D. J. Pharm. Pharmacol. 1970, 22, 261264. (8) Lopez-Berestein, G. Antimicrob. Agents Chemother. 1987, 31, 675-678. (9) Gabizon, A. Liposomes as a drug delivery system in cancer chemotherapy In Drug carrier systems; Roerdink, F. H. D., Kron, A. M., Eds.; John Wiley & Sons, Ltd.: Chichester, U.K.
S0743-7463(97)00353-3 CCC: $14.00
therapeutic index observed with several antimonials,10,11 imunomodulators,12,13 antifungal agents,14,15 and antibiotics.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 controlled 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 vesicles22 composed of dioctadecyldimethylammonium 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 adhesion 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(10) New, R. R. C.; Chance, M. L.; Heath, S. J. Antimicrob. Chemother. 1981, 8, 371-381. (11) New, R. R. C.; Chance, M. L.; Thomas, S. C.; Peters, W. Nature (London) 1978, 272, 55-56. (12) Fraser-Smith, E. B.; Eppstein, D. A.; Larsen, M. A.; Matheus, T. R. Infect. Immun. 1983, 39,172-178. (13) Lopez-Berestein, G.; Bodey, G. P.; Frankel, L. S.; Mehta, K. J. Clin. Oncol. 1987, 5, 310-317. (14) Lopez-Berestein, G.; Hopfer, R. L.; Mehta, R.; Mehta, K.; Hersh, E. M.; Juliano, R. L. J. Infect. Dis. 1984, 150, 278-283. (15) Lopez-Berestein, G.; MacQueen, T.; Mehta, K. Cancer Drug Delivery 1985, 1, 37-42. (16) Backer-Woodenberg, I. A. J. M.; Lokerse, A. F.; Vink-van Der Berg, J. C.; Roerdink, F. H.; Michel, M. F. Antimicrob. Agents Chemother. 1986, 30, 295-300. (17) Bourventre, P. F.; Gregoriadis. G. Antimicrob. Agents Chemother. 1978, 16, 1049-1051. (18) Al Awadhi, H.; Stokes, G. V.; Reich, M. J. Antimicrob. Chemother. 1992, 30, 303-311. (19) Bakker-Woudenberg, I. A. J. M.; Lokerse, A. F.; ten Kate, M. T.; Melissen, P. M. B.; van Vianen, W.; van Etten, E. W. M. Eur. J. Clin. Microbiol. Infect. Dis. 1993, 12, 61-67. (20) Desiderio, J. V.; Campbell, S. G. J. Infect. Dis. 1983, 148, 563570. (21) Hand, W. L.; King-Thompson, N. L. Antimicrob. Agents Chemother. 1986, 29, 135-140. (22) Carmona-Ribeiro, A. M. Chem. Soc. Rev. 1992, 21, 209-214. (23) Ta´pias, G. N.; Sicchierolli, S. M.; Mamizuka, E. M.; CarmonaRibeiro, A. M. Langmuir 1994, 10, 3461-3465.
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gregated cells at much smaller cell concentrations than those required to attain rapid bacteria flocculation.24 Furthermore, the interaction of DODAB vesicles with both normal and transformed mammalian cells has been recently evaluated suggesting a strong necessity of a deeper and systematic analysis of DODAB’s full potential to interact with drugs, antigenic proteins, or genes, delivering them to cells at low ionic strength.25 In this work, we further characterize DODAB vesicles as bactericides by determining dose and time effects on four different bacteria species of clinical importance. Some mechanistic aspects of the bacteria/vesicle interaction leading to cell death are also investigated. Although death does involve interaction with the external bacterial wall, cell lysis or vesicle rupture does not occur, suggesting that damage to membrane proteins rather than membrane lysis is a possibility. The fact that these cationic vesicles in the rigid gel state do not disintegrate upon adsorption to the bacterial cell surface might be of considerable significance to deliverance by endocytosis of drugs inside the vesicle aqueous compartment. Materials and Methods Organisms and Culture Conditions. Isolated E. coli, Salmonella typhimurium, Staphylococcus aureus, or Pseudomonas aeruginosa were grown overnight at 37 °C in a tube containing 15 mL of trypticase soy broth (Difco Laboratories, Detroit, MI). Thereafter, the culture was transferred to 150 mL of the same nutritive broth and incubated on a shaker (150 rpm) at 37 °C for 5 h. The turbid suspension thus obtained was centrifuged at 8000 rpm for 15 min, and the pellet was resuspended in phosphate buffer saline, (PBS), pH 7.2. This last procedure was repeated twice before finally resuspending the pellet in isotonic 0.264 M D-glucose. This final step was necessary because the small synthetic amphiphile vesicles (SV) used throughout aggregate and fuse in the presence of low monovalent salt concentrations (above 5-10 mM NaCl).26 Bacteria or vesicles were always diluted using a 0.264 M D-glucose solution. Bacteria number densities were determined from agar plating and cfu counting, turbidity against a Macfarland scale, or a correlation established between bacteria number density (obtained by cfu counting) and turbidity at 400 nm. Chemicals. Dioctadecyldimethylammonium bromide 99.9% pure (DODAB) was obtained from Fluka Chemie AG (Switzerland) and used as such without further purification. Dioctadecyldimethylammonium chloride (DODAC) was obtained by ion exchange from DODAB using AMBERLYST A-26 from E. Merck (Darmstadt, Germany) in the chloride form as previously described.27 All other reagents were analytical grade and were used without further purification. Water was Milli-Q quality. Vesicle Preparation. Small unilamellar DODAB vesicles (SV) with an 86 nm mean diameter28 were prepared by ultrasonic dispersion with a tip in a 0.264 M D-glucose solution.29 SVs were centrifuged at 10 000 g for 1 h at 15 °C to precipitate multilamellar liposomes and titanium particles ejected from the titanium probe during sonication. The supernatant containing the unilamellar vesicles was used within 1 h of the preparation. Large unilamellar DODAC vesicles (LV) with 250 nm mean diameter were prepared by vaporization of a chloroformic DODAC solution as previously described.29 DODAB or DODAC concentrations were determined by microtitration.30 Microelectrophoresis of Escherichia coli Ghosts. Ghosts of the isolated external membrane of E. coli were obtained using (24) Sicchierolli, S. M.; Mamizuka, E. M.; Carmona-Ribeiro, A. M. Langmuir 1995, 11, 2938-2943. (25) Carmona-Ribeiro, A. M.; Ortis, F.; Schumacher, R. I.; Armelin, M. C. S. Langmuir 1997, 13, 2215-2218. (26) Carmona-Ribeiro, A. M.; Chaimovich, H. Biophys. J. 1986, 50, 621-626. (27) Lessa, M. M.; Carmona-Ribeiro, A. M. J. Colloid Interface Sci. 1996, 181, 166-171. (28) Carmona-Ribeiro, A. M.; Midmore, B. R. J. Phys. Chem. 1992, 96, 3542-3547. (29) Carmona-Ribeiro, A. M.; Chaimovich, H. Biochim. Biophys. Acta 1983, 733, 172-179. (30) Schales, O.; Schales, S. S. J. Biol. Chem. 1941, 140, 879-883.
Martins et al. a method (procedure II) previously described by Henning et al.31 Ghosts and vesicles were allowed to interact for 1 h at room temperature before the electrophoretic mobility (EM) measurements were performed as a function of DODAB concentration in the mixtures. Mobilities were measured using a Rank Brothers microelectrophoresis apparatus with a flat cell at 25 °C. The sample to be measured was placed into the electrophoresis cell, electrodes were connected, and a voltage of 60 V was applied across the cell. Velocities of individual ghosts over a given tracking distance were recorded, as was the direction of ghost movement. Average velocities were calculated from data on at least 20 individual ghosts. EM was calculated according to the equation EM ) cm(u/V)(1/t), where u is the distance over which the particle is tracked (micrometers), cm is the interelectrodes distance (7.27 cm), V is the voltage applied ((60 V), and t is the average time in seconds required to track one particle a given distance u. Determination of Vesicle Rupture. Vesicle rupture was assessed by marking the internal aqueous compartment of large DODAC vesicles (LV) with [14C]-sucrose as previously described29 and measuring radioactivity leakage from the vesicles upon interaction with the bacteria at 2 × 108 bacteria/mL. Leakage was detected from equilibrium dialysis of the vesicle/bacteria mixture using a equilibrium dialysis chamber with two compartments (a and b) separated by a cellulose membrane. Three experiments, A, B, and C, were performed. In A, compartment a contained 1 mL of [14C]-sucrose plus a 0.264 M D-glucose solution. In B, compartment a contained 1 mL of a 2 mM DODAC LV dispersion in 0.264 M D-glucose with its intravesicular compartment containing [14C]-sucrose. In C, compartment a contained a mixture of E. coli and marked DODAC LV (2 × 108 bacteria/mL and 2 mM DODAC, respectively). In all three experiments, compartment b contained 1 mL of a 0.264 M D-glucose solution. Counts per minute (cpm) in compartments a and b were determined as a function of time from 50 µL aliquots. Vesicle rupture was detected from the appearance of radioactivity in compartment b. Determination of Leakage of Intracellular Contents. DODAB-induced leakage of intracellular phosphorylated compounds was used as a measurement of cell lysis. Sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB), recognized for their ability to induce phospholipid micellization and lysis of cell membranes, were also included in the experiment for comparison. One milliliter aliquots of 6.9 × 109 bacteria/mL in 0.264 M D-glucose were centrifuged (5000g/15 min/15 °C) so that the cells forming a pellet in the bottom of the centrifugation tube were thereafter resuspended with 1.0 mL of synthetic amphiphile dispersions (0.1-100 mM DODAB, CTAB or SDS). The bacteria/amphiphile mixture was vortexed and left to interact for 0.5 h. As a control, one aliquot of the bacterial suspension (1 mL, 6.9 × 109 bacteria/mL) was pelleted and resuspended with 1.0 mL of a 0.264 M D-glucose solution (without amphiphile) following the same procedure. Thereafter, all mixtures with the exception of the control were pelleted again and the inorganic phosphate concentration was determined in the supernatants and in the control suspension as previously described.32 Bacterial cell lysis is assumed to be linearly related to the percentage of leakage of the phosphorylated compounds appearing in the supernatant and is calculated from the ratio between the inorganic phosphate concentration in the supernatant and that in the control as % phosphate leakage ) 100 [Pi(supernatant)/Pi (control)]. Viability Assays. Colony-forming units (cfu) counting was obtained as a function of DODAB concentration at 2 h of interaction time between bacteria and vesicles or as a function of time at 5 µM DODAB. Vesicles and cells interacted at high bacteria number densities (higher than 107 bacteria/mL) before mixtures were diluted (1:104 or 1:105 ) and 0.1 mL of each diluted mixture was spread on an agar plate. After spreading, plates were incubated for 24 h at 37 °C. Cfu counts were made using a colony counter. (31) Henning, U.; Rehn, K.; Hoehn, B. Proc. Natl. Acad. Sci. 1973, 70, 2033-2036. (32) Rouser, G.; Fleischer, S.; Yamamoto, A. Lipids 1970, 5, 594596.
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Figure 1. Electrophoretic mobility of E. coli ghosts as a function of DODAB concentration in the bacteria/vesicles mixtures.
Figure 3. Leakage of phosphorylated compounds (%) as a function of surfactant type and concentration.
Figure 4. Cell viability (%) as a function of DODAB concentration at 2 h of interaction time between DODAB SV and bacteria. About 100% viability was obtained for each control sample containing bacteria only (in the absence of DODAB). Bacteria number densities in the interacting mixtures are given in the legend. Before plating 0.1 mL in agar, interaction mixtures were diluted 1:104 (S. Aureus) or 1:105 (other species). This yields 490 (9), 730 (b), 500 (2), and 1050 (1) cells per agar plate.
Figure 2. Counts per minute (cpm) in compartments a and b of a dialysis chamber as a function of time. Compartment a contains a radioactively labeled D-glucose solution (A), a 2 mM DODAC LV dispersion where vesicles had their inner compartment radioactively labeled (B), a mixture of DODAC LV (2 mM final concentration), and E. coli (2 × 108 bacteria/mL) (C).
Results and Discussion Figure 1 shows the electrophoretic mobility of E. coli ghosts as a function of DODAB concentration at two different number densities for the ghosts. Mobilities increase as a function of DODAB concentration yielding curves that are very similar to those previously obtained for entire E. coli cells considering the differences in cell concentration.24 The interaction between E. coli and DODAC LV was also investigated from the vesicle rupture point of view. In Figure 2A, it is shown that the [14C]-sucrose initially placed in hemichamber a diffuses across the cellulose
membrane until equilibrium is achieved with half of the initial amount of radioctivity on each side. In Figure 2B, it is shown that DODACs LV containing [14C]-sucrose do not let their contents leak so that the radioactivity in each compartments remains practically equal to the initial level. In Figure 2C, the interaction between E. coli and DODAC LV in compartment a leads to fairly equal initial and final amounts of radioactivity, showing the absence of bacteriainduced vesicle rupture. Cell lysis effects induced by SDS, CTAB, and DODAB are shown for E. coli and S. aureus (Figure 3). Percent leakage of phosphorylated compounds from the cells increases as a function of amphiphile concentration (C) both for CTAB and SDS. In comparison to surfactants that assemble as micelles, the bilayer-forming DODAB does not cause significant cell lysis. This is consistent with the absence of vesicle rupture shown in Figure 2 because cell lysis by the vesicles would require formation of mixed micelles containing surfactant and lipids from the cell membrane. In fact, DODAB is not a micellizing agent and DODAB molecules do not assemble as micelles but as bilayer vesicles.22 The effect of DODAB concentration (C) on cell viability for four different model microorganisms from agar plating and cfu counting is in Figure 4. For a 2 h interaction with 1 mM DODAB, no viable cell was detected for the four species tested. One should notice the very large cell
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Figure 5. Cell viability (%) as a function of interaction time between bacteria and DODAB SV at 5 µM (b). Cell concentrations in the interaction mixtures are 1.00 × 108, 1.65 × 108, 0.64 × 10 8, and 1.11 × 108 cells/mL for S. aureus (A), S. typhimurium (B), E. coli (C), and P. aeruginosa (D), respectively. Plating on agar (0.1 mL) was done after a 1:104 dilution of the mixture, yielding 1000, 1650, 640, and 1110 cells plated, respectively. Viabilities determined for control mixtures (in the absence of DODAB) are also shown (O).
number density in the interaction mixtures with DODAB (Figure 4). Although DODAB-induced bacteria flocculation was previously described at these high number densities, dilution of 1:104 or 1:105 before plating causes cells to disaggregate, as visualized from optical microscopy (not shown). Thus, underestimation of viability due to DODAB-induced flocculation and clustering of several viable cells to yield only one viable count is not a possibility. The effect of time on cell viability was obtained at a fixed DODAB concentration (Figure 5). At 5 µM DODAB, depending on the species and the cell concentration in the interacting mixture, the time required for attaining less than 20 cfu per plate varies between 1 and 5 h (Figure 5). One should notice that a comparison between different species cannot be performed if cell number densities are not the same or, at least, very similar. For instance, cfu from 1000 S. aureus cells can be compared to cfu from 1110 P. aeruginosa cells. The latter die first in the presence of DODAB (Figure 5A and D). However, further comparisons between sensitivities to DODAB depend on adjusting cell quantity on interacting mixtures and on plates to the same value. In summary, DODAB seems to act differently in comparison with other cationics that disrupt the cell membrane. The mechanism of DODAB bactericidal action possibly involves damage to protein function at the bacterial external wall level, where vesicles do adhere (Figure 1) without vesicle rupture (Figure 2) or cell lysis (Figure 3).
The aim of this study was threefold: (1) reconfirm the bactericidal effect of cationic vesicles previously described for E. coli;24 (2) observe whether the bactericidal action described for E. coli could be generalized to other bacteria of clinical importance; (3) investigate the mechanism of death in comparison to that of other surfactants that form micelles. We have shown that bacterial death caused by DODAB vesicles occurs for the four different bacteria species tested (Figures 4 and 5). This is particularly important from the point of view of antibiotics incorporation in DODAB or DODAC liposomes, since conventional cationic liposomes usually encapsulate more efficiently antibiotics such as amikacin, netilmicin, and tobramycin.33 A possible synergistic action between antimicrobial liposomes such as DODAB SV or DODAC LV and antibiotics may become useful in clinics. Also bacterial cell lysis was demonstrated to be absent both for a Gram-negative bacterium (E. coli) and for a Gram-positive (S. aureus) bacterium, in clear contrast to observations by other authors that detected lytic effects induced by micelleforming quaternary ammonium surfactants.1,7 Thus, bilayer-forming surfactants such as DODAB and DODAC clearly behave differently and must be causing death via a mechanism that does not include cell lysis. Consistently, interaction between bacteria and large DODAC vesicles did not induce leakage of intravesicle contents (Figure 2). Vesicle fusion with the cell membrane would necessarily (33) Omri, A.; Ravaoarinoro, M.; Possion, M. J. Antimicrob. Chemother. 1995, 36, 631-639.
Cationic Vesicles as Bactericides
lead to leakage. Therefore, lipids from the cell membrane and DODAC molecules from the vesicle do not seem to mix, as expected for lipids that are in different physical states (lipids from the cell membrane are in the liquidcrystalline, more fluid state whereas DODAB and DODAC are in the gel, rigid state). In fact, DODAC and DODAB are not micellizing agents capable of disrupting cell membranes. Furthermore, we have previously quantified the adsorbed DODAB or DODAC amount on the surface of the bacterial cells.23 At limiting adsorption, a palisade of vesicles, i.e., a monolayer of adjacent vesicles, was demonstrated to be adhered on the cell surface.23 The palisade model adequately explained the amphiphile amount adsorbed on the bacterial surface. Thus, mere vesicle adhesion to the cells seems to have devastating effects that lead to cell death. A strong possibility is damage to the function of the membrane proteins in charge of transport through the bacterial cell wall in the case of the Gram-negative bacteria. Would death involve obstruction of porins by entire, nondisrupted vesicles? This is an open question presently being addressed by us.
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The results presented in this work establish DODAB or DODAC liposomes by themselves as effective bactericides, in contrast to the usually innocuous effect of conventional liposomes used as drug carriers. Regarding the potential clinical utility of DODAB as a bactericide, the present work conducted in isotonic D-glucose solution may be at odds with any clinical use since under physiological conditions the ionic strength is much higher. Nevertheless, lipid mixtures containing DODAB could well have an improved colloidal stability without loss of the DODAB bactericidal effect. This possibility is being investigated in our laboratory. Acknowledgment. L.M.S.M. thanks FAPESP for an undergraduate fellowship. FAPESP and CNPq are gratefully acknowledged for research grants 96/0704-0 and 520186/96.6, respectively. LA970353K
