8230
J. Phys. Chem. B 2001, 105, 8230-8236
Interactions between Cationic Vesicles and Candida albicans M. T. N. Campanha˜ , E. M. Mamizuka, and A. M. Carmona-Ribeiro* Departamento de Bioquı´mica, Instituto de Quı´mica, UniVersidade de Sa˜ o Paulo, CP 26077, CEP 05513-970 Sa˜ o Paulo SP, Brazil ReceiVed: September 16, 2000; In Final Form: June 20, 2001
Dioctadecyldimethylammonium bromide (DODAB), a bilayer-forming synthetic amphiphile with antibacterial properties, also affects viability of Candida albicans. For C. albicans, simultaneous determination of cell viability and electrophoretic mobility as a function of DODAB concentration yields a very good correlation between cell surface charge and cell viability. Upon increasing DODAB concentration, the cell surface charge decreases and changes its sign to yield positively charged cells. However, in contrast to the DODAB bactericidal property, the amphiphile effect on fungus over 1 h of interaction time is only fungistatic at 1 mM DODAB and ca. 106 cells/mL. Nevertheless, solubilization of fungicides in DODAB dispersions prepared by sonication causes complete loss of cell viability. Amphotericin B (AB) or miconazole (M) solubilization in the DODAB bilayer leads to 100% cell death, though the drug and DODAB effects are independent. Cell and DODAB bilayer membranes compete for AB or M solubilization with the amphiphile bilayer controlling drug release to the cell membrane. 0.1 mM DODAB plus AB or M concentration of 2 and 20 µg/mL, respectively, yielded 0% of cell viability for C. albicans (2 × 106 cells/mL) over an interaction time of 24 h. The cationic liposomes by themselves yielded ca. 20% viability under similar conditions. The full potential of these cytotoxic cationic bilayers to control release and cytotoxicity of drugs remains hitherto unexplored.
Introduction Some synthetic bilayer-forming amphiphiles have found many different uses in strategically applied areas.1,2 In particular, cationic liposomes of dioctadecyldimethylammonium (DODA) salts have been sucessfully employed to interact with negatively charged surfaces or biomolecules such as prokaryotic3 or eukaryotic cells,4 antigenic proteins,5 nucleotides,6 nucleic acids,7,8 synthetic polymers and latex,9,10 and mineral surfaces.11,12 Since 1994, the cytotoxicity of DODAB dispersions and other cationic liposomes against several bacteria species and cultured mammalian cells has been described,3,4,13-18 as was their ability to solubilize amphotericin B (AB) and other hydrophobic drugs such as miconazol (MCZ).16 UV-visible spectra for waterinsoluble drugs plus particle sizing experiments clearly demonstrated previously that the insoluble drug crystals present in pure water completely disappeared upon addition of the DODAB dispersion obtained by sonication with tip (Vieira and CarmonaRibeiro, submitted for publication). In the present work, DODAB antifungal action against Candida albicans is evaluated from the effect of DODAB vesicles on fungus viability both by themselves or containing AB or MCZ. Effects of DODAB concentration, interaction time, and liposomal incorporation of antifungal drugs on cell viability of C. albicans are determined. Presently and previously obtained results3,4,13-16 are gathered and compared from the point of view of differential cytotoxicity of DODAB liposomes toward pathogenic microorganisms and cultured mammalian cells. Mammalian cells4 are much more resistant to DODAB-induced cell death than are bacteria3 or fungi (this work). Cultured mammalian fibroblasts remain 50% viable at 1.0 mM DODAB,4 whereas bacteria3,13-16 or fungi (this work) exhibit 50% of viability over the micromolar range * To whom correspondence should be addressed. FAX: 055 11 3815 5579. E-mail:
[email protected] of DODAB concentrations (5-28 µM DODAB). Furthermore, in this work the independence of DODAB and drug action is demonstrated from the competition between fungus and DODAB membranes for drug solubilization with retardation of drug action induced by DODAB at large DODAB concentrations. Materials and Methods Chemicals. Dioctadecyldimethylammonium bromide (DODAB) was from Sigma and used as received. Amphotericin B (750-13A) was obtained from Squibb and used as such without further purification. Amphotericin B concentration in the amphotericin powder from Squibb was determined from its optical spectra in methanol as previously described.19 The absorptivity for AB in methanol at 405 nm was reported to be E1% 1cm ) 1812, i.e., 1 g of AB solubilized in 100 mL of methanol would yield an absorbance at 405 nm equal to 1812 absorbance units. 10 mg of the AB powder used in this work was solubilized in 100 mL of methanol. From this stock solution, six other methanol solutions containing 0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 µg/mL of AB were prepared. Ultraviolet spectra for these solutions were recorded, and the absorbance at 405 nm was plotted against AB concentration yielding a straight line as expected from the Lambert-Beer law. From these data, the AB powder used in this work yielded E1% 1cm ) 1699 ( 65, a parameter that is equivalent to a molar absorptivity of 156 952 ( 6025 M-1 cm-1. This indicates a percentage of drug present in the powder equal to 93 ( 4%. The nominal drug potency from Squibb is 95.5%. All other reagents were of analytical grade and used without further purification. Water was Milli-Q quality. Preparation of DODAB Dispersions. Ultrasonic dispersion with tip in a 0.264 M D-glucose solution followed by centrifugation (12100 g/1 h/15 °C) to precipitate multilamellar liposomes
10.1021/jp003315+ CCC: $20.00 © 2001 American Chemical Society Published on Web 08/04/2001
Cationic Vesicle Interactions with C. albicans and titanium particles ejected from the titanium probe during sonication2 was the procedure used to produce a mixture of DODAB vesicles and bilayer fragments (SV) with 86 nm mean diameter.2 The supernatant containing the DODAB aggregates was used within 1 h of the preparation. DODAB concentrations were determined by microtitration.20 It is important to emphasize that vesicles were always prepared in the absence of the antifungal drugs to be used in the experiments. Organism and Culture Conditions. Candida albicans ATCC 90028 isolated in 4% dextrose Sabouraud agar was plated in 2% dextrose of the same agar. After a period of 24 h growth in agar plates, one or two colonies were transferred to 30 mL of Sabouraud broth, and turbidity (625 nm) was adjusted to 0.5 of a MacFarland scale. Thereafter, the suspension was incubated in a shaker (30 °C/160 rpm/6 h), centrifuged (12100 g/15 min), and the pellet resuspended in a 0.264 M D-glucose solution. This last washing procedure was repeated twice before adjusting turbidity to a final cell concentration of ca. (1-2) × 106 cells/ mL or ca. (1-2) × 107 cells/mL. Cell Viability Assays in the Presence of DODAB. Cell viability was determined as a function of the following variables: (1) DODAB concentration at a fixed interaction time of 1 h for two different cell concentrations (1.7 × 106 and 1.2 × 107 cells/mL); (2) interaction time between cells and DODAB vesicles at 0.13 or 1.3 mM DODAB concentration and 1.0 × 106 cells/mL; (3) interaction time between cells and drug carrying DODAB vesicles at 0.11 and 1.1 mM DODAB plus 2 or 20 µg/mL of AB or MCZ, respectively. In the first and in the second case, 0.1 mL of a DODAB small-vesicle dispersion (0.01-10.00 mM DODAB in D-glucose 0.264 M) was mixed with 0.9 mL of a C. albicans suspension (also in D-glucose 0.264 M), previously adjusted (625 nm) to ca. 1.3 × 106 cells/mL or 1.2 × 106 cells/mL. For determining the DODAB concentration effect, the mixtures interacted at 25 °C over 1 h before being diluted and plated (0.1 mL) on Sabouraud/2% dextrose agar plates. Each mixture was plated in quadruplicate, and viability (%) was expressed as a mean value with an error bar corresponding to the mean standard deviation. A similar protocol was followed for determining time effects on cell viability at a fixed cell concentration. Microelectrophoresis of C. albicans in the Presence of the Cationic Liposomes. DODAB dispersions over a range of DODAB concentrations and C. albicans were mixed to yield a final cell concentration of 1.0 × 106 cells/mL, allowed to interact for 1 h at room temperature, and placed in a flat cell to determine fungi electrophoretic mobility (EM). One should notice that the measurements were done precisely at the same experimental conditions used to obtain the viability curves for C. albicans (1.7 × 106 cells/mL) as a function of DODAB concentration in the mixtures. Mobilities were determined using a Rank Brothers microelectrophoresis apparatus with a flat cell at 25 °C. The sample to be measured was placed into the electrophoresis glass cell, electrodes were connected, and a voltage of (60 V was applied across the glass cell. Velocities of individual fungi over a given tracking distance were recorded, as was direction of fungi movement. Average velocities were calculated from data on at least 20 individual fungi. 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 (6.606 cm), V is the voltage applied ((60 V), and t is the average time in seconds required to track one cell a given distance u. Cell Viability Assays in the Presence of DODAB plus Drugs. (a) Determination of Drug Concentration Effects on Cell
J. Phys. Chem. B, Vol. 105, No. 34, 2001 8231 Viability. A 1 mg/mL of AB stock solution in 1:1 v/v dimethyl sulfoxide (DMSO)/methanol was prepared. 1, 2, 4, 6, 8, and 10 µL of the stock solution were added to 1 mL of a DODAB dispersion to yield AB concentrations equal to 1, 2, 4, 6, 8, and 10 µg/mL. 0.5 mL of small DODAB vesicles dispersion at ca. 2 mM DODAB in D-glucose 0.264 M containing AB over the 0-10 µg/mL range of AB concentration was mixed with 0.5 mL of a C. albicans suspension (also in D-glucose 0.264 M) previously adjusted to ca. 2 × 106 cells/mL. The mixtures interacted at 25 °C over 1 h before being diluted (1:103) and plated (0.1 mL) on Sabouraud/2% dextrose agar plates. As a control for the solvent effect, AB and DODAB powders, in the complete absence of solvent, were dispersed by sonication, diluted, and tested against the fungus following a similar protocol yielding essentially the same results as those obtained in the presence of traces of solvent (0.5% solvent, at most, for the largest drug concentration). A similar experiment was done for mixtures that interacted over 24 h. In this case, another AB stock solution containing 0.1 mg/mL was used to prepare 0.2-10 µg/mL of AB in the DODAB dispersion. 1, 2, 4, 6, 8, and 10 µL of 0.1 mg/mL or 1 mg/mL AB in DMSO/methanol were added to 1.0 mL of 2.0 mM DODAB dispersion. Thereafter, 0.5 mL of the fungi suspension was mixed with 0.5 mL of the liposomal AB dispersion and left interacting over 24 h. For miconazole, 10.0, 2.0, 0.2, 0.02, and 0.002 mg/mL stock solutions in methanol were prepared. After that, 20 µL of the 10 mg/mL stock solution was added to 1.0 mL of 2 mM DODAB dispersion to obtain a 200 µg/mL solution. 10 µL of 2.0, 0.2, 0.02, and 0.002 mg/mL stock solutions was added to 1 mL of 2 mM DODAB dispersion. 0.5 mL of the small DODAB vesicle dispersion at ca. 2 mM DODAB containing MCZ over the 0-200 µg/mL range of MCZ concentration was mixed with 0.5 mL of a C. albicans suspension previously adjusted to ca. 2 × 106 cells/mL. The mixtures interacted over 1 or 24 h before being diluted (1:103) and plated (0.1 mL) on Sabouraud/ 2% dextrose agar plates. Each mixture was plated in quadruplicate, and viability (%) was expressed as a mean value with an error bar corresponding to the mean standard deviation. Similarly, a control for the solvent effect was performed by sonicating the DODAB/MCZ dispersion in complete absence of solvent. Also in this case, C. albicans viability did not sense the trace amounts of solvent used in the experiments. (b) Determination of Interaction Time Effects at a GiVen DODAB and Drug Concentration. AB stock solution (0.5 mg/ mL) in 1:1 v/v dimethyl sulfoxide (DMSO)/methanol was prepared. 8 µL of the stock solution was added to 1 mL of a DODAB dispersion prepared as above to yield a drug concentration of 4 µg/mL. 0.5 mL of this DODAB dispersion containing the drug was added to 0.5 mL of the C. albicans suspension to yield a final drug concentration of 2 µg/mL. For miconazole, a M stock solution (5 mg/mL) in methanol was prepared. 8 µL of the MCZ stock solution was added to 1 mL of a DODAB dispersion prepared as above to yield a drug concentration of 40 µg/mL. 0.5 mL of this DODAB dispersion containing the drug was added to 0.5 mL of the C. albicans suspension to yield a final drug concentration of 20 µg/mL. Thereafter, these drug containing DODAB dispersions were assayed to establish their effects on cell viability as a function of interaction time between C. albicans cells and drug containing DODAB dispersions. For the experiments with liposomal amphotericin B or miconazole, 0.5 mL of a DODAB dispersion at ca. 2 or 0.2
8232 J. Phys. Chem. B, Vol. 105, No. 34, 2001
Campanha˜ et al.
Figure 1. Cell viability (%) as a function of DODAB concentration at 1 h of interaction time between DODAB SV and C. albicans. For controls containing fungus only (in absence of DODAB), 100% viability was obtained. Before plating 0.1 mL on agar, interaction mixtures were diluted 1:1000 (0) or 1:10000 (b).
Figure 2. Cell viability (%) as a function of interaction time between fungus and DODAB SV at two different final DODAB concentrations. Viable cell concentration for each interaction mixture is 1.3 × 106 CFU/ mL. Before plating 0.1 mL on agar, interaction mixtures were diluted 1:1000.
mM DODAB containing AB or MCZ at 2 or 20 µg/mL, respectively, was mixed with 0.5 mL of a C. albicans suspension previously adjusted to ca. 1 × 106 cells/mL. Drug carrying DODAB dispersions and fungi interacted up to 72 h with aliquots being diluted (1:103 or 1:104) and plated (0.1 mL) on Sabouraud/ 2% dextrose agar plates over a range of interaction times. Similarly, each mixture was plated in quadruplicate and viability (%) was expressed as a mean value with an error bar corresponding to the mean standard deviation. Mean viability (%) was plotted as a function of interaction time.
TABLE 1: Differential Cytotoxicity of Cationic DODAB Liposomesa
Results 1. The Antifungal Action of Cationic DODAB Liposomes against C. albicans. Figure 1 shows the DODAB concentration effect on viability of C. albicans at two different cell concentrations. At 1.7 × 106 CFU/mL, viability steeply decreased at ca. 10 µM DODAB up to 18% at a few tenths of micromolar of DODAB concentration. At 1.2 × 107 CFU/mL, a cell concentration ca. 10 times higher than the previous one, the smallest viability attained was 50%. This cytotoxicity of 50% is much lower than the 80% cytotoxicity expected from the vesicles/ cells ratio and might be due not only to the increase in total cell surface area available for DODAB adsorption but also to DODAB-induced cell aggregation. On basis of our previous experience with bacteria and mammalian cells, DODAB-induced cell aggregation, a phenomenon kinetically dependent on cell concentration, might also be occurring at the highest cell concentration.3,4,13,14,16 This phenomenon would be rapid at 1.2 × 107 cells/mL so that cell surfaces for those cells in the middle of cell aggregates would not be reached by DODAB vesicles, which would explain the unexpectedly low cytotoxicity obtained at the higher cell concentrations. Figure 2 shows cell viability as a function of interaction time between vesicles and C. albicans cells at 1 × 106 cells/mL. Over the first hour of interaction, there was a steep decrease of viability that remained constant thereafter at 18 or 10% of viability for 0.1 or 1.0 mM DODAB, respectively. This first set of results (Figures 1 and 2) indicated that DODAB vesicles, over the range of concentrations and interaction times tested, cannot be considered as fungicides i.e., as agents that cause 99.9% of viability loss. The present results for fungi show that
cell type normal Balb-c 3T3 (clone A31) mouse fibroblasts SV40-transformed SVT2 mouse fibroblasts C. albicans E. coli S. typhimurium P. aeruginosa S. aureus
viable cell concentration (cells/mL)
DODAB concentration for 50% survival (mM)
ref
104
1.000
4
104
1.000
4
0.010 0.028 0.010 0.005 0.006
this work 3 and 15 15 15 15
2 × 106 2 × 107 2 × 107 3 × 107 3 × 107
a Interaction time between small DODAB vesicles and cells was fixed at 1 h. The data were taken from the references quoted in the last column.
they are more resistant to the antimicrobial properties of DODAB than were the bacteria previously tested.3,15 However, mammalian cells were previously shown4 to be still more resistant than the fungi. This could be clearly seen from the comparison of the DODAB amount required to cause 50% of cell death shown in Table 1. DODAB indeed exhibits differential cytotoxicity, an important property for therapeutic uses. Although the present work dealt only with C. albicans, it was timely to gather all (present and past) results on DODAB cytotoxicity in a single table (Table 1). Figure 3 shows the correlation between C. albicans electrophoretic mobility and cell viability as a function of DODAB concentration. Electrophoretic mobility for the population of C. albicans cells (1 × 106 cells/mL) was zero at ca. 10 µM DODAB, a concentration where about 50% of the cells were positively charged and dead whereas the other 50% were negatively charged and alive (Figure 3). A similar correlation between sign on the cell surface and life or death was previously observed also for Gram-positive or Gram-negative bacteria15 and for mammalian cells.4 The occurrence of a positive charge on the cell surface led to death, possibly due to damage of vital transport functions by membrane proteins. Consistently, absence
Cationic Vesicle Interactions with C. albicans
J. Phys. Chem. B, Vol. 105, No. 34, 2001 8233
Figure 3. Eletrophoretic mobility (EM) (b) and cell viability (9) for C. albicans (1 × 106 CFU/mL) as a function of DODAB concentration. Fungus and vesicles in the mixtures interacted for 1h before dilution and plating or EM measurements.
of lysis for the cell membrane was previously reported as was absence of cationic vesicle rupture.3 In favor of the interpretation that positively charged cells were dead is the previous observation that only positively charged cells were observed under the dark field optical microscope of the electrophoretic mobility apparatus at 0% of viability for bacteria.15 2. Controlled Release of Amphotericin B or Miconazole to C. albicans Using DODAB Dispersions Prepared by Sonication. The effect of 2 µg/mL amphotericin B either solubilized in its solvent or in cationic DODAB liposomes against C. albicans is shown in Figure 4. From 0.1 micrograms per mL of AB, the drug was considered as a fungicide.24 The drug concentration used (2 µg/mL) was chosen because it was expected to kill the fungus under standard experimental conditions.21 Although our experiments were not carried out under these standard conditions (liposomes and cells are throughout in D-glucose 0.264 M only), this fungicide concentration for cells in rich media was used as a reference. A criticism at this point might have been raised regarding toxicity of the solvent or solvent mixture used to solubilize the drugs. These solvents were indeed toxic to the cells, but only 5 microliters of solvent (DMSO/methanol for AB or methanol for MCZ) was added per milliliter of cell suspension. This is only 0.5% of the total volume, so that the solvent effect on viability is expected to be basically negligible or very small against the large biocidal effects that were measured in this work. In Figure 4B, the DODAB concentration of 1.1 mM was tested in the presence and absence of 2 µg/mL of amphotericin B. In this case, drug dilution in the liposomal phase led to a minimum of 16% viability, a percentile also obtained for 1.1 mM DODAB without drug. At 1.1 mM DODAB, the drug had no detectable effect on the cells over all the time interaction range of observation. The liposomal phase was competing with the fungi cell membrane for drug solubilization. At large DODAB amounts, as is 1.1 mM DODAB, the liposomal phase won the competition and the drug was not delivered to the cell membrane. Therefore, a reduction of 10 times in DODAB concentration should have improved competition of the cell membrane and drug delivery to the fungus, and it did as seen from Figure 4A. At 0.11 mM DODAB, there was an increased drug deliverance to the fungus cell membrane with cell viability falling to 0% in the presence of drug (Figure 4A). At 0 mM DODAB, C. albicans viability in the presence of the drug alone
Figure 4. Cell viability (%) as a function of interaction time between fungus and liposomal amphotericin B (AB) for two different DODAB concentrations: 0.11 (A) and 1.1 mM DODAB (B). Viable cells concentration is ca. 1.3 × 106 CFU/mL in both cases. Two controls were performed for effects on viability of the drug alone ([) or DODAB alone (9).
(solubilized in its organic solvent mixture) yielded 0% (Figure 4A and B). Once added to the water solution where the fungi were, the drug rapidly went to the cell membrane of the fungi, effectively causing its death (Figure 4). The drug in its best solvent was apparently very effective in killing the fungi in Vitro, though solvent toxicity was expected to hamper its use as the drug solubilizer in therapy. One should notice that the DODAB dispersion prepared by sonication was as efficient as the best drug solvents to solubilize the drugs.16 Thus, DODAB was fulfilling at least three important functions: (1) drug solubilization; (2) retardation of drug action; (3) display of an independent antimicrobial action. The mode of action of amphotericin B had repeatedly been associated with its specific complexation with ergosterol, a component of the fungi cell membrane absent in bacteria.22,23 The DODAB cationic bilayer in its rigid gel state did not need ergosterol to solubilize and carry amphotericin B.16 The affinity of AB for some sites in the DODAB bilayer seemed to be so high that DODAB retarded AB delivery to the fungus cell membrane. To confirm the results obtained for amphotericin B, another hydrophobic antifungal drug of current use was tested (Figure 5). Miconazole is not as potent as is amphotericin B. Its inhibitory action against C. albicans usually occurs over a drug concentration range that is ca. 10 times higher than the one reported for amphotericin B.24 Miconazole carried by the cationic DODAB bilayers became quite effective as a fungicide at 20 µg/mL (Figure 5), a final miconazole concentration previously reported to be only fungistatic.24,25 The difference in cell viability obtained for drug alone in its organic solvent
8234 J. Phys. Chem. B, Vol. 105, No. 34, 2001
Figure 5. Cell viability (%) as a function of interaction time between fungus and liposomal miconazole (M) at 0.11 mM DODAB. Viable cells concentration is 1.3 × 106 CFU/mL. Two controls were performed for effects on viability of the drug alone (2) or DODAB alone (9).
and drug plus liposome could not be considered significant within the limits of the experimental error (Figure 5). However, the fact remains that 0% of viability was obtained for both drugs while formulated as a combination of drug and cationic DODAB in the absence of drug solvent (Figures 4 and 5). Furthermore, the controlled drug release by DODAB was interesting for decreasing drug cytotoxicity against mammalian cells. At this point the discrepancy between the expected fungistatic effect of miconazole and its observed fungicidal effect (at 48 and 72 h, in Figure 5) should be discussed. The fungistatic effect of miconazole as reported from the literature was obtained under rich-medium conditions where the drug may adsorb and/or complex with several components in the mixture thereby being sequestered by the medium and becoming less available to adsorb and act on the cell surface. Typical rich medium conditions are 150 mM ionic strength and pH 7.4, whereas the present work was all done at pH of pure water (6.3) and ionic strength close to zero. This could explain why miconazole in the present experimental conditions became more effective against the fungus. At pH 6.3 and low ionic strenght the drug is positively charged (its apparent pK is 6.5) and electrostatically attracted to the oppositely charged cell. This electrostatic attraction is at maximum due to the ionic strength close to zero in the present experiments. 3. Additive but Independent Antimicrobial Action for DODAB Dispersions and Fungicides Incorporated in the Cationic Bilayers. Minimal inhibitory concentrations (MIC) of amphotericin B and miconazole for C. albicans are 1.9 and 0.016-100 µg/mL for Candida sp, respectively, for a drug/ fungus interaction of 48 h in a rich medium21,24 (this broad range for MIC being ascribed to the expected dose variability effective against different yeast species). These MIC values were taken into account in order to define the drug concentration range for testing the DODAB/drug effect on C. albicans in D-glucose 0.264 M. The rationale for choosing a drug concentration range including the MIC values was the eventual detection of a killing effect for the drug inside the cationic bilayer that was absent for the drug alone. In fact, cell viability decreased as a function of liposomal amphotericin B concentration attaining a minimum equal to zero at 3-4 µg/mL of amphotericin B and 24 h of interaction or at 5 µg/mL of amphotericin B and 1 h of
Campanha˜ et al.
Figure 6. Cell viability (%) for C. albicans as a function of amphotericin B concentration (µg/mL) at 1 h of interaction time and 1.1 × 106 cells/mL (0) or 24 h of interaction time and 2.9 × 106 cells/ mL (b). The drug was previously incorporated in small DODAB vesicles (final DODAB concentration equal to 1 mM). The control containing DODAB vesicles only (without drug) is shown for the interaction over 1 h (0) or 24 h (O). Before plating 0.1 mL in agar, interaction mixtures were diluted 1:103. This yields 110 (0) and 290 cells plated per agar plate (b).
Figure 7. Cell viability (%) for C. albicans as a function of miconazole concentration (µg/mL) at 1 h of interaction time and 1.5 × 106 cells/ mL (0) or 24 h of interaction time and 3.5 × 106 cells/mL (b). The drug was incorporated in DODAB small vesicles (final DODAB concentration equal to 1 mM). The control containing DODAB vesicles only (without drug) is shown for the interaction over 1 h or 24 h (dashed or dotted lines). Before plating 0.1 mL in agar, interaction mixtures were diluted 1:104. This yields 150 (0) and 350 cells plated per agar plate (b).
interaction (Figure 6). Considering that assays used for determining MIC values (drug concentration range for inhibiting growth) always used at least 48 h of interaction time between drug and fungus, the fungicidal action obtained in Figure 6 at 1 and 24 h of interaction could be interpreted as being due to the addition of the drug and the antimicrobial DODAB effect. For miconazole, a drug less effective than amphotericin B, complete absence of survival was obtained only at 100 µg/mL of liposomal miconazole and 1 h of interaction time (Figure 7). One should notice that this dose in absence of the cationic
Cationic Vesicle Interactions with C. albicans DODAB liposomes was expected to be only fungistatic and not fungicidal. There was also a combined action between the cationic liposomes and miconazole, which became even more evident from the very short interaction time of 1 h used for the experiment in Figure 7. In Figures 6 and 7, one should notice that 1.0 mM DODAB alone (in absence of drug) yielded 28% (1h) or 18% of survival (24 h). This indicated a much less effective action for DODAB on fungi than on bacteria, since at 1 mM DODAB no survival at all was previously observed for four different bacteria species of clinical importance.3,15 Discussion The interaction between DODAB and cells in 0.264 M D-glucose solution (chosen to preserve isotonicity between the internal and the external cell environment) caused a well-defined cytotoxicity over the same DODAB concentration range that changed the cell surface charge from negative33 to positive (Figure 3). This cytotoxicity was much higher for bacteria and fungi than for mammalian cells in culture. The challenge will be preservation of this differential cytotoxicity at physiological conditions. This work has not shown definitely that the drug is delivered to the cell by the cationic liposome. There is also the possibility that the lipid is, in fact, just a solvent and that the drug partitions out of the liposome into the aqueous phase, from which it enters the cells. However, amphotericin B is poorly soluble (10-7 M) in water where it occurs as aggregates.25-27 Admittedly, we cannot specify a deliverance mechanism at this point; eventually liposomes and drug might have separate effects on the cells that can be added in any way to efficiently and differentially kill C. albicans cells. From a more general point of view, the antimicrobial properties of the quaternary nitrogen moiety in amphiphiles have been systematically observed in the literature since 1935.28,29 The killing of C. albicans by a series of single chained amphiphilic quaternary ammonium compounds (QACs) with different hydrocarbon chain lengths was closely related to the binding of the compounds to the cells and damage of the cell membranes.30 Generally, the mechanism of action for single chained quaternary ammonium salts involves the micellizing effect of the single-chained surfactant on the cell membrane that leads to its disruption. DODAB, however, is a doublechained amphiphile that forms bilayers instead of micelles. As expected, its mechanism of action on the cells turns out to be different. We have already demonstrated for E. coli cells that large DODAB vesicles neither disintegrate upon interaction with the cell wall nor cause cell disruption.3 This correlates nicely with the extremely low cmc for DODAB and its bilayer existence in water dispersion as bilayer vesicles with absence of micelles or individual DODAB monomers in solution. The retarding effect of DODAB on drug activity was previously observed also for other amphiphiles. In the presence of palmitoyl mannose, the cytotoxicity of AB is decreased toward both fungal and mammalian cells; while its fungistatic potential is increased, its inflammatory properties are conserved and its acute toxicity is significantly diminished.31 These effects were explained by the formation of a complex between AB and the sugar ester that impedes the interaction of the drug with either serum components or cell membrane constituents. The effect of Myrj 59 (a polyoxyethyleneglycol derivative of stearic acid) was AB solubilization, abolishment of its haemolytic activity, and preservation of the in-vitro antifungal activity of the drug.32 An excellent discussion about the competition between target cells and carrier for the drug is also available from Brajtburg
J. Phys. Chem. B, Vol. 105, No. 34, 2001 8235 and Bolard.27 AB is a drug of choice for the treatment of most systemic fungal infections, which is marketed under the trademark Fungizone, an AB-deoxycholate complex suitable for intravenous administration. This association AB/deoxycholate is relatively weak; therefore, dissociation occurs in the blood. The drug itself interacts with both mammalian and fungal cell membranes to damage cells, but the greater susceptibility of fungal cells to its effects forms the basis for its clinical use.27 The ability of the drug to form stable complexes with lipids has allowed the development of new formulations of AB based on this property. Several lipid-based formulations of the drug, which are more selective in damaging fungal or parasitic cells than mammalian cells and some of which also have a better therapeutic index than Fungizone, have been developed.27 In vitro investigations have led to the conclusion that the increase in selectivity observed is due to the selective transfer of AmB from lipid complexes to fungal cells or to the higher thermodynamic stability of lipid formulations. Association with lipids modulates AmB binding to lipoproteins in vivo, thus influencing tissue distribution and toxicity. For example, lipid complexes of AmB can be internalized by macrophages, and the macrophages then serve as a reservoir for the drug. Furthermore, stable AB-lipid complexes are much less toxic to the host than Fungizone and can therefore be administered in higher doses.27 The results of this work suggest a new possible formulation using DODAB to carry AB (from now on named: DOD/AB). What is so special or new about our DOD/AB formulation? Basically its new insight is joining the quaternary ammonium antimicrobial effect and advantages of liposomal formulations to modify stability and drug biodistribution. Like all noninnocuous cationic surfactants with the quaternary ammonium moiety, DODAB exhibits antimicrobial properties.3,13-16 Like all innocuous phospholipids that form liposomes, DODAB assembles in aqueous solutions as bilayer vesicles that do not disintegrate upon interaction with cells3 and are, as all liposomes, potential carriers for hydro- or liposoluble drugs.1,2,16,17,25 The utility of drug formulations based on DODAB assemblies remains hitherto unexplored but is presently one of the main lines of research in our lab. Acknowledgment. M.T.N.C. thanks FAPESP for a fellowship. FAPESP and CNPq are gratefully acknowledged for research grants. References and Notes (1) Fendler, J. H. Acc. Chem. Res. 1980, 13, 7. (2) Carmona-Ribeiro, A. M. Chem. Soc. ReV. 1992, 21, 209. (3) Martins, L. M. S.; Mamizuka, E. M.; Carmona-Ribeiro, A. M. Langmuir 1997, 13, 5583. (4) Carmona-Ribeiro, A. M.; Ortis, F.; Schumacher, R. I.; Armelin, M. C. S. Langmuir 1997, 13, 2215. (5) Tsuruta, L. R.; Quintilio, W.; Costa, M. H. B.; Carmona-Ribeiro, A. M. J. Lipid Res. 1997, 38, 2003. (6) Kikuchi, I. S.; Viviani, W.; Carmona-Ribeiro, A. M. J. Phys. Chem. A 1999, 103, 8050. (7) Kikuchi, I. S.; Carmona-Ribeiro, A. M. J. Phys. Chem. B 2000, 104, 2829. (8) Behr, J. P. Acc. Chem. Res. 1993, 26, 274. (9) Carmona-Ribeiro, A. M.; Midmore, B. R. Langmuir 1992, 8, 801. (10) Carmona-Ribeiro, A. M.; Lessa, M. M. Colloid Surf. A 1999, 153, 355. (11) Rapuano, R.; Carmona-Ribeiro, A. M. J. Colloid Interface Sci. 1997, 193, 104. (12) Rapuano, R.; Carmona-Ribeiro, A. M. J. Colloid Interface Sci. 2000, 226, 299. (13) Ta´pias, G. N.; Sicchierolli, S. M.; Mamizuka, E. M.; CarmonaRibeiro, A. M. Langmuir 1994, 10, 3461.
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