a SmithKline Beecham Pharmaceuticals, Drug Delivery group, Harlow CM17 5AW, Essex b Pharmaceutical Sciences Institute, Aston University, Birmingham B4 7ET, UK
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Abstract |
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Introduction |
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Pulmonary clearance can be reduced by encapsulation of antimicrobials within phospholipid liposomes. 10 ,11 ,12 ,13 Liposomal encapsulation of polymyxin B has proved successful in extending the pulmonary residence time in adult rats. 14 In addition to changing the pulmonary pharmacokinetics of polymyxin B, liposomal encapsulation may also influence antibacterial efficacy. 15 Encapsulation has been shown to enhance the activity of piperacillin against staphylococci, and of tobramycin and ticarcillin against resistant strains of Pseudomonas aeruginosa. 16 ,17 Phospholipid composition may play an important role in determining interaction between liposomes and bacteria, with positively charged lipids mediating electrostatic attraction between liposomes and negatively charged bacterial membranes. 18
The aim of this study was to investigate the efficacy of a range of liposome-encapsulated polymyxin B formulations against a clinical isolate of P. aeruginosa grown in chemically defined media. Liposomal polymyxin B formulations were characterized by determination of drug loading, particle size (by laser light diffraction) and zeta potential by laser Doppler velocimetry. MICs were measured and antimicrobial bactericidal studies performed, comparing the activity of liposomal polymyxin B with equivalent concentrations of non-entrapped polymyxin B.
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Materials and methods |
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Unless specified otherwise, chemicals and reagents were supplied by BDH Chemicals Ltd (Poole, UK), Sigma Chemical Co. (Poole, UK) or Fisons (Loughborough, UK) and were of Analar grade or equivalent. Polymyxin B sulphate was purchased from Sigma. The activity was 7730 IU/mg. Phospholipids were purchased from Lipid Products, South Nutfield, UK. All phospholipids were stored in chloroform/methanol at -20°C. Tritiated polymyxin B ([3H] polymyxin B) was obtained from NEN Life Science Products (Hounslow, UK) and had a specific activity of 39.59 MBq/mL (1.07 mCi/mg).
Organism and culture maintenance
The organism used in these studies was P. aeruginosa NCTC 6750 (PA 6750). It was maintained at 4°C on nutrient agar (Lab M Ltd, Bury, UK) plates. Long-term storage was at -70°C in chemically defined medium (CDM12; see below).
Chemically defined medium (CDM12)
PA 6750 was grown in CDM12 liquid medium (48 mM glucose, 0.74 mM KCl, 0.60 mM NaCl, 48 mM (NH)4SO4, 48 mM MgSO4·7H2O, 60 mM 3-(N-morpholino) propane-sulphonic acid (MOPS), 3.84 mM K2HPO4·3H2O, double-distilled water to 1 L) and shaken on a rotating incubator (New Brunswick G10) at 37°C. The pH was adjusted to 7.8 using 1 M NaOH and the medium was autoclaved at 121°C for 20 min. Glucose and K2HPO4·3H2O were autoclaved separately to prevent caramelization and precipitation respectively. Omission of iron from the medium ensured iron-depleted growth.
Preparation of liposomal polymyxin B for antimicrobial bactericidal assay and MIC studies
Dehydrationrehydration vesicle (DRV) preparations of liposomal polymyxin B were prepared as previously described. 19 Briefly, lipids were added to a round-bottomed flask and sufficient chloroform added to obtain a thin film. The lipid mixture was dried by rotary evaporation under reduced pressure, and solvent traces were removed by drying under a nitrogen stream. The resulting thin film was redispersed with phosphate-buffered saline (PBS) pH 7.4 by gentle agitation for incubation for 30 min at a temperature above the phospholipid transition temperature. The resulting preparation was sonicated to clarity using a probe sonicator (probe diameter 14 mm, amplitude 18,000; Soniprep 150, MSE, Crowley, UK), for 10 cycles of 60 s on and 30 s off. Heat generated during sonication was dispersed by a bath of iced water. The resulting suspension of small unilamellar vesicles (SUV) was centrifuged (Beckmann JII; Beckmann Instruments Ltd, High Wycombe, UK) at 400g for 10 min to remove large lipid aggregates and titanium particles. An aliquot of SUV (60 µmol of total lipid) was mixed with an aliquot of polymyxin B solution (9 mg polymyxin B) and the mixture frozen at -70°C. After freezing, the preparation was freeze-dried for 18 h (Edwards Modulyo Freeze-drier; Edwards High Vacuum Ltd, Crowley, UK). The preparation was rehydrated with a volume of distilled water equivalent to one tenth of the total volume of SUV used, at a temperature greater than the phospholipid transition temperature. Rehydration was aided by vortex mixing (30 s) and by incubation with shaking at the required temperature for 30 min. This procedure was repeated for two further rehydration steps using 1.8 mL and 4.0 mL of PBS pH 7.4.
Liposomes were separated from non-entrapped drug by centrifugation (18,000gfor 30 min, Beckman JII). This was repeated using 20 mL of buffer. The final pellet was resuspended by vortexing with 1 mL of PBS and making up to 5 mL with buffer. Liposomes were stored at 4°C until required. Encapsulations were determined by scintillation counting of [3H] polymyxin B in the retained supernatant washes and in the final liposome suspension.
Zeta potential analysis of liposomal polymyxin B formulations
Zeta potentials of empty and loaded vesicles were measured by laser Doppler velocimetry to investigate the effect of drug loading on liposome surface charge. Liposome dispersion (20 µL, c. 0.3 µmol of lipid) was added to 10 mL of 0.02 M diphosphate buffer (Na2HPO4·2H2O 3.598 g/L; KH2PO4 2.72 g/L; pH 7.4) and filtered through a filter with 0.2 µm pores; zeta potentials were then measured at 25°C ± 0.1°C using a Zetamaster instrument (Malvern Instruments Ltd, Malvern, UK). Machine operation was periodically checked using a latex standard with defined electrophoretic mobility.
Laser diffraction sizing of liposomal polymyxin B formulations
The Malvern Mastersizer E (Malvern Instruments) was used to size liposomal suspensions by laser light diffraction. A quantity (200 µL, c. 3 µmol lipid) of liposome dispersion was vortex-mixed with 10 mL of filtered buffer (0.22 µm pore-size polycarbonate filter; Millipore, UK) and sized. Liposome diameters are reported as the equivalent volume mean D ± S.D.
Antimicrobial bactericidal assay
The growth of an iron-depleted batch culture of PA6750 was followed by the measurement of optical density at 470 nm. Cells in the early logarithmic phase of growth were used for bactericidal studies. Tubes containing appropriate concentrations of non-entrapped polymyxin B and liposomal polymyxin B (to give final concentrations of 0.1 mg/L and 0.3 mg/L respectively) in CDM salt solution were prepared aseptically in triplicate and prewarmed at 37°C. Control tubes were prepared by omitting antimicrobial solutions and using an appropriate volume of CDM12 salt solution. Additional control tubes, containing empty liposomes at the same lipid concentration as loaded vesicles, were also prepared. Cultures of PA6750 were used at optical densities between 0.04 and 0.08, and then standardized by dilution with CDM12 salts solution to an OD470nm of 0.04. Aliquots of this suspension (100 µL) were added to the antimicrobial solutions (4.9 mL) to give the desired cell density (1x 10 6 cfu/mL) and incubated at 37°C for 1 h.
To determine cell numbers after exposure to free and liposomal polymyxin B, colony counts were determined using the spread plate method. 20 The treated cells and controls were diluted 1:9 and 1:99 in Letheen broth (Difco Laboratories, Detroit, MI, USA) and 100 µL samples plated on to predried nutrient agar in triplicate. The dilutions and inoculum were chosen to produce 30300 colonies per plate. Plates were incubated at 37°C overnight and the resulting colonies were enumerated using a colony counter. The viable count for the original suspension was established from the mean number of colony-forming units (cfu) from triplicate sets of plates and multiplied by the dilution factor (cfu/mL). The relative reductions in cfu were compared and expressed as the percentage surviving fraction for each test solution.
Determination of the MICs of polymyxin B and liposomal polymyxin B
Aliquots of double-strength CDM (2.4 mL) were dispensed into test-tubes with 0.5 mL glucose (0.4 M). Polymyxin B/liposomal polymyxin B and distilled water were added in a 2 mL volume to give the desired concentration in a final volume of 5 mL and finally a 100 µL inoculum (diluted in CDM to give 1 x 106 cfu/mL) from an overnight culture was added. The final concentrations (in 5 mL) of both free and liposomal polymyxin B were 0.10.8 mg/L. The tubes were vortexed and incubated at 37°C on a shaker for 18 h. The tubes were examined for growth and the MIC defined as the lowest concentration of antibiotic that inhibited the development of visible growth.
The quantity of drug released from liposomes during the period of MIC testing was determined by incubating a control tube containing CDM salts solution with liposomal polymyxin B in the absence of inoculum. After 18 h incubation at 37°C, samples were taken and free drug separated using Microcon 100 microconcentrators (Amicon, Gloucester, UK). Release was expressed as the percentage of drug initially encapsulated, and this value was used to determine the concentration of free polymyxin B in solution.
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Results |
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DRV were prepared using phospholipid compositions which would give nominally neutral vesicles (egg phosphatidylcholine (EPC)), negatively charged vesicles (EPC:dicetyl phosphate (DCP) 9:1) and positively charged vesicles (EPC:stearylamine (SA) 9:1). The entrapment of polymyxin B within these formulations is shown in Figure 1. Similar loadings were achieved with EPC (45.41% ± 0.51%, n = 3) and EPC:DCP vesicles (50.81% ± 0.79%). Positively charged vesicles had a significantly lower entrapment (31.92% ± 2.08%). Electrophoretic mobility measurements in 0.02 M diphosphate pH 7.4 illustrate the different surface characteristics of the liposome preparations used (Figure 2). Empty liposomes composed solely of EPC had a zeta potential of -0.3 ±0.3 mV. The inclusion of a 10% molar ratio of SA resulted in a positive potential of 12.8 ± 1.1 mV. Liposomes containing the negative amphiphile, DCP, had a strongly negative zeta potential of -54.9 ± 2.7 mV. When the zeta potential of liposomes with entrapped polymyxin B was measured, the positivity of all preparations increased. Liposome diameter was determined by laser diffraction and the mean volumes of all loaded preparations were found to be similar (EPC, 5.05 ± 2.31 µm; EPC:DCP, 5.98 ± 4.02 µm; EPC:SA, 5.70 ± 4.36 µm).
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The antimicrobial activity of polymyxin B was assessed using two concentrations; 0.1 and 0.3 mg/L. Figure 3 shows the relative differences in bactericidal activity expressed as the percentage surviving fraction for non-entrapped polymyxin B and liposomal polymyxin B at a concentration of 0.3 mg/L. Incubation of empty vesicles at lipid concentrations equivalent to loaded vesicles gave results similar to those of CDM salt control tubes, indicating that the phospholipids used had no effect on growth. At 0.3 mg/L, positively charged and negatively charged liposomes produced a greater reduction in cell numbers than neutral liposomes.
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The entrapment of polymyxin B within DRV used for MIC studies is shown in the Table. The MIC for free polymyxin B against stationary phase cells of PA6750, grown in iron-depleted CDM12, was found to be 0.10.2 mg/L. The MICs of neutral EPC-based liposomes varied when expressed as the total concentration of polymyxin B. However, when the effective free polymyxin B concentration was calculated using the percentage of drug released, the MIC for all neutral EPC-based formulations was similar (c. 0.10.2 mg/L). Only EPC:SA liposomes had a lower MIC (0.0440.066 mg/L). The MICs observed with DSPC-based vesicles were lower than those of free drug.
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Discussion |
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As P. aeruginosa cells are negatively charged (-11.4 ± 2.9 mV) in PBS, 23 the enhanced activity of positively charged vesicles is due to increased cell association through attractive electrostatic interactions. The enhanced activity of negatively charged liposomes is unlikely to be the result of electrostatic interactions. It is possible that the surface polarity of DCP-containing vesicles resulted in increased surface hydrophilicity, which would facilitate association with hydrophilic PA6750 cells. The technique of hydrophobic interaction chromatography has been applied to characterize the surface hydrophilicity of liposome formulations, but characterization of loaded EPC:DCP vesicles gave ambiguous results for this parameter. 24 Alternatively, increased surface association of polycationic polymyxin B with the anionic headgroup of DCP may increase the availability of polymyxin B at the liposome surface to interact with the target lipopolysaccharide (LPS) molecules on the bacterial surface.
A number of previous studies have investigated the effect of liposome encapsulation on bactericidal activity in vitro. Negatively charged tobramycin-loaded liposomes were more active than free drug, at sub-MIC levels, against a clinical isolate of pseudomonas. It has been suggested that there was sufficient cellliposome attraction to overcome any electrostatic repulsion, but a mechanism was not proposed. 25 These findings are similar to those seen with negatively charged polymyxin B formulations at a dose of 0.3 mg/L. Charge effects were also reported by Song & Jones 26 who described a strong association of cationic liposomes with films of adsorbed streptocooci and staphylococci. It was found that the number of liposomes (100140 nm in diameter) per bacterium ranged from 1000 to 3000 depending on the bacterial strain. Positive charge was also shown to increase the activity of ciprofloxacin-loaded liposomes against a clinical isolate of P. aeruginosa. 23 Variation of lipid acyl chain and cholesterol content had little effect on cell-association. Electrostatic attraction was proposed as the mechanism for increased association of these vesicles. Entrapped aminoglycosides and penicillins have been shown to be effective against strains of E. coli and P. aeruginosa resistant to the free antibiotics. 13 ,15 ,17 The proposed mechanism of enhancement included protection of drug from enzymatic degradation and facilitation of diffusion of liposome-associated drug across the bacterial envelope. Translocation of phospholipids between the outer and inner membranes of Salmonella typhimurium occurs after direct fusion of phospholipid vesicles. 27 Sekeri-Pataryas et al. 28 showed that liposomes containing entrapped penicillin could overcome the cell wall barrier of resistant P. aeruginosa and effectively deliver the antibiotic to the cell. Liposomebacterial fusion has been demonstrated with the delivery of a liposome-entrapped enzyme, horse-radish peroxidase, to bacterial cells. 29 Diffusion facilitated by phospholipid vesicles was proposed to deliver this molecule, which is normally too large to diffuse across the outer membrane of bacterial cells.
Other studies have demonstrated the importance of the availability of entrapped drug in determining antibiotic activity in vitro. The inactivity of encapsulated chloramphenicol and streptomycin against E. coli in broth culture was noted by Stevenson et al. 30 The vesicles were loaded with sufficient antibiotic which, if present as free drug in solution, would have inhibited bacterial growth. Similarly, ampicillin-loaded liposomes failed to inhibit growth of Listeria monocytogenes in culture. 31 Gentamicin and ceftazidime-loaded stealth vesicles had low bactericidal activity against Klebsiella pneumoniae in culture, yet were effective against infection in vivo. 32 In such cases the release of entrapped antibiotic would determine activity in vitro. It is likely that for each particular bacteriumliposomal antibiotic interaction, a number of the above mechanisms are appropriate and generalizations regarding in-vitro activity cannot be made.
In the case of liposomal polymyxin B, entrapment within phospholipid vesicles does not result in a reduction in activity at the two concentrations tested. As liposomal polymyxin B did not show enhanced activity relative to free drug at 0.1 mg/L, it would appear that liposome-facilitated diffusion of polymyxin B does not occur. The results achieved with liposomal polymyxin B must be qualified in terms of the fraction released and, therefore, the amount of polymyxin B available for bactericidal activity. Although release experiments were not conducted in CDM salt solution, release in PBS pH 7.4 indicated that only a fraction of the entrapped contents was released at later stages of the procedure (generally <30%after 4 h). The activity of liposomal polymyxin B is, therefore, likely to be attributable to release of polymyxin B at or near the cell surface, which would expose bacterial cells to a transiently high polymyxin B concentration. Protection of polymyxin B from enzymatic degradation is unlikely to enhance activity significantly, as the drug is not extensively hydrolysed by bacterial enzymes. Although liposomal polymyxin B did not possess significantly enhanced antimicrobial bactericidal activity, the increased pulmonary residence time of this formulation should provide a more effective treatment of infection in vivo than that achievable with free drug. 33
The MIC observed with liposomal formulations clearly depended on the availability of free drug in solution. For all neutral EPC-based formulations, the MIC values were similar to that of free drug, suggesting that liposomal encapsulation did not promote diffusion/delivery to bacterial cells. The lower MIC value of EPC:SA liposomes indicates greater association of EPC:SA vesicles with bacterial cells, which would result in increased polymyxin B released in the cell microenvironment. The increased activity of EPC:SA encapsulated polymyxin B in the MIC assay is consistent with the greater bactericidal activity of this formulation relative to neutral vesicles. A previous study has demonstrated enhanced activity of aminoglycosides against P. aeruginosa when entrapped within cationic liposomes. 34 In the current study, the MICs observed with DSPC-based vesicles were lower than those of free drug. This is surprising given that release from this type of vesicle was less than that seen with EPC-based formulations and resulted in lower (sub-MIC) concentrations of free drug in the incubation medium. Previous studies have noted that the acyl chain composition of vesicles has little effect on association with P. aeruginosa cells. 23 As the entrapment of DSPC-based vesicles was usually lower than that of comparable EPC-based vesicles, a larger lipid dose was needed to achieve the equivalent polymyxin B dose. Thus the higher ratio of vesicles to cells increases the possibility of liposomecell interaction and subsequent release of polymyxin B within the immediate vicinity of bacterial membranes. Alternatively, the altered membrane fluidity of DSPC-based vesicles may promote adsorption of cells to liposomal surfaces.
The antimicrobial efficacy of liposomal polymyxin B was investigated using cell-kill and MIC determinations. In both cases, efficacy of polymyxin B was at least comparable to that of free drug, indicating that the DRV encapsulation process was not detrimental to antimicrobial activity. Numerous studies have reported an enhancement of antimicrobial activity by liposomal encapsulation through putative mechanisms of increased diffusion or protection from enzymatic degradation. The current study has shown that the antimicrobial activity of polymyxin B is not enhanced in this way, and differences between liposome compositions are likely to be the result of either electrostatic interactions (for positively charged vesicles) or increased vesicle/cell ratios (DSPC containing formulations). The in-vivo efficacy of liposomal polymyxin B is likely to be determined by the pulmonary clearance of free and entrapped polymyxin B, and the rate of polymyxin B release from the liposomal carrier.
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Acknowledgments |
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Notes |
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References |
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Received 23 February 1998; returned 27 April 1998; revised 17 June 1998; accepted 17 August 1998