1 The Novel Drug&Vaccine Delivery Systems Facility, Department of Chemistry and Biochemistry, Laurentian University, 935 Ramsey Lake Rd, Sudbury, Ontario, P3E 2C6, Canada; 2 The University of Texas Health Center, Department of Medicine, 11937 US Highway 271, Tyler, Texas 75708, USA
Received 5 August 2004; returned 22 October 2004; revised 27 October 2004; accepted 28 October 2004
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Abstract |
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Methods: Gentamicin was encapsulated into liposomes with different lipid compositions (1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-distearoyl-sn-glycero-3-phosphocholine) and cholesterol in the molar ratio of 2:1 by sonication. The in vitro stability of liposome-encapsulated gentamicin was studied over a 48 h period at 4 and 37°C in PBS and at 37°C in pooled plasma. The MICs of free and liposomal gentamicin for clinical isolates of P. aeruginosa were assessed by broth dilution.
Results: The encapsulation efficiency of all liposomal preparations was 4%5.18% of the initial amount of the drug in solution. The liposomes retained 60%70% of the encapsulated gentamicin for 48 h when they were incubated in normal human pooled plasma or PBS at 4 or 37°C. The MICs of liposomal gentamicin for all clinical isolates of P. aeruginosa were lower than the MICs of free gentamicin. Importantly, liposomal gentamicin altered the susceptibilities of these clinical isolates from gentamicin resistant to either intermediate or susceptible.
Conclusions: Taken together, these data indicate that liposomal gentamicin formulations could be more effective than the free drug in controlling pulmonary infections due to P. aeruginosa.
Keywords: antibiotic delivery , lung infection , stability
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Introduction |
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The present study was undertaken to evaluate encapsulation efficiency, in vitro stability and antibacterial activity of our newly developed liposomal gentamicin formulations against resistant strains of P. aeruginosa.
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Materials and methods |
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Encapsulation efficiency was calculated as the percentage of gentamicin incorporated in liposomes relative to the initial total amount of gentamicin in solution. The loading capacity was calculated as the amount of gentamicin incorporated in liposomes relative to the content of total lipid. The concentration of gentamicin incorporated into liposomes was measured by agar diffusion assay, using a laboratory strain of Staphylococcus aureus (ATCC 29213) as an indicator organism.5
The in vitro stability of liposome-encapsulated gentamicin was determined in PBS, pH 7.2 or in normal human pooled plasma at 4 or 37°C with mild agitation. After incubation periods of 0.25, 0.5, 1, 3, 6, 12, 24 and 48 h, samples were removed and centrifuged (18300 g for 15 min at 4°C) to remove the liposomal gentamicin. The free gentamicin concentrations in the supernatants were determined by agar diffusion assay.5 Antibiotic release was expressed as a percentage of liposomal retention of the initially encapsulated gentamicin.
We studied the antibacterial effect of these formulations on non-mucoid (PA-1, PA-489122 and M-57192R) and mucoid (PA-489121, PA-48913 and M-26250) strains of P. aeruginosa isolated from sputum of pulmonary infected cystic fibrosis patients at the Memorial Hospital (Sudbury, ON, Canada). Laboratory strains of S. aureus (ATCC 29213) and P. aeruginosa (ATCC 27853) were used as test organisms as well as reference strains for quality control. The MICs of free and liposomal gentamicin for clinical isolates of P. aeruginosa were determined as previously described.5
The data are expressed as means ± S.E.M. of three independent experiments. Comparisons were made by paired Student's t-test and P 0.05 was considered significant. For multiple comparisons within and between groups, ANOVA with the two-tailed Dunnett's post-test analysis was used.
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Results |
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All liposomal preparations retained >70% of the initially encapsulated gentamicin up to 48 h in PBS. Generally, however, liposomes incubated at 4°C retained more antibiotic than those incubated at 37°C. For instance, the liposomes composed of DPPC-CHOL (DPPCcholesterol) retained significantly more antibiotic at 4°C than those stored at 37°C (89.3% ± 1.3% versus 80.6% ± 1.2%, P < 0.0001) at the end of a 48 h experimental period. We also compared the drug release profile of different liposomal formulations and found that the liposomes composed of DPPC-CHOL retained more antibiotic than that of DMPC-CHOL at 4°C (89.3% ± 1.3% versus 77.3% ± 1.1%, P < 0.0003) in 48 h. Likewise, the liposomes composed of DPPC-CHOL retained more antibiotic than that of DSPC-CHOL (89.3% ± 1.3% versus 84.1% ± 1.2%, P < 0.005) at the end of a 48 h experimental period. However, the drug-release data obtained at 37°C did not indicate any significant difference in the lipid compositions.
To mimic physiological conditions, we determined the drug-release kinetics of liposomal gentamicin incubated in normal human pooled plasma at 37°C (Figure 1). All liposomal formulations released 40% of the encapsulated drug in 48 h. Although we did not detect a significant difference in the release kinetics of gentamicin in plasma in relation to different liposomal formulations, we found that the liposomes incubated in PBS at 37°C retained significantly more antibiotic than when they were incubated in plasma over a period of 48 h. For instance, the liposomes composed of DPPC-CHOL retained 80.6% ± 1.2% of initially encapsulated drug at 37°C in PBS, compared with 64.1% ± 4.3% (P < 0.0001) of the same formulation in plasma.
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Discussion |
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Our drug-release data reported here clearly indicate a significant improvement in gentamicin retention by our formulations, regardless of the temperature. The contributing factors include our choice of lipids, the ratio of lipid to cholesterol (2:1, molar ratio) and the chemical nature of the antibiotic, gentamicin. We found that liposomes composed of DPPC-CHOL retained more antibiotic in PBS at 4°C than liposomes composed of DSPC-CHOL or DMPC-CHOL. Although at present we do not know why the DPPC-CHOL formula increases the stability of these liposomes in PBS, other studies echo similar findings. It has been reported that the DPPC-encapsulated paclitaxel, an anti-tumour agent, is more stable in PBS than the liposomes composed of DMPC or DSPC.9 These authors argued that paclitaxel incorporation increases the phase-transition temperature of the lipid vesicles and that this broadening effect was greatest for DPPC, hence leading to a more stable compound. Although our formulations incubated in normal human pooled plasma released 20% more antibiotic than those incubated in PBS at 37°C, they performed well when compared with liposomes composed of egg phosphatidylcholine.10
All clinical strains of P. aeruginosa used in this study are considered resistant (NCCLS) to gentamicin (MIC16 mg/L). Our formulations, however, significantly enhanced the susceptibilities of these organisms to gentamicin, from highly resistant to either intermediate (MIC
8 mg/L) or susceptible (MIC
4 mg/L) to this antimicrobial agent. Liposomes may protect the encapsulated drug from the action of bacterial enzymes as well as facilitating its diffusion across the bacterial envelope. To the best of our knowledge, however, this is the first report on liposomal formulations that enhance gentamicin antibacterial activity against gentamicin-resistant clinical strains of P. aeruginosa. We are investigating the mechanism by which these formulations enhance gentamicin activity against the resistant strains of P. aeruginosa.
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Acknowledgements |
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References |
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