Liposome-mediated gentamicin delivery: development and activity against resistant strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients

Clement Mugabe1, Ali O. Azghani2 and Abdelwahab Omri1,*

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


* Corresponding author. Tel: +1-705-675-1151 ext. 2190; Fax: +1-705-675-4844; Email: aomri{at}nickel.laurentian.ca

Received 5 August 2004; returned 22 October 2004; revised 27 October 2004; accepted 28 October 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Chronic pulmonary infection by Pseudomonas aeruginosa in cystic fibrosis patients is virtually impossible to eradicate by means of existing free antibiotics. We sought to assess the antibacterial activities of liposomal gentamicin against clinical isolates of P. aeruginosa.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pseudomonas aeruginosa is a major cause of nosocomial infections and accounts for ~95% of deaths in the cystic fibrosis population.1 A major reason for its prominence as a pathogen is its high intrinsic resistance to most available antibiotics.2 A drug-delivery system that could reduce antibiotic toxicity while increasing the therapeutic indices is of great interest, and liposome-encapsulated antimicrobial agents can provide these benefits.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We encapsulated gentamicin into liposomes with different lipid compositions [1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)] and cholesterol in the molar ratio of 2:1 by a sonication method, as previously described.3 The average particle size and the polydispersity index4 were determined by laser light scattering with the use of a NICOMP 270/autodilute Submicron Particle Sizer (Santa Barbara, CA, USA).

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-48912–2 and M-57192R) and mucoid (PA-48912–1, 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The average diameter of liposome-encapsulated gentamicin with different lipid compositions varied from 408 ± 28 to 418 ± 21 nm, with no significant difference between various liposomal preparations. The polydispersity index [the measurement of homogeneity of dispersion, ranging from 0.0 (homogeneous) to 1.0 (heterogeneous)] for the size distribution of liposomal formulations ranged from 0.59 ± 0.009 to 0.74 ± 0.007. Encapsulation efficiencies of gentamicin in all three types of liposomes were 4%–5.18% of the initial amount of the drug in solution. The loading capacity of gentamicin, however, was formula-dependent and varied significantly (P < 0.001) from 26.7 ± 1.3 to 34.5 ± 1.5 µg/µmol.

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 (DPPC–cholesterol) 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.



View larger version (15K):
[in this window]
[in a new window]
 
Figure 1. Gentamicin retention in liposomes of various lipid compositions in human plasma. Liposome-encapsulated gentamicin composed of DMPC-CHOL (filled circles), DPPC-CHOL (filled triangles), DSPC-CHOL (filled squares) were incubated at 37°C in normal human pooled plasma with mild agitation. At the times indicated, plasma samples were removed (centrifugation at 18300 g for 15 min at 4°C) and gentamicin concentrations in the supernatants were determined by agar diffusion microbiological assay. Results are means±S.E.M. of three separate experiments. All formulations retained a comparable amount of gentamicin at the end of a 48 h assay period.

 
The MICs of all three liposomal gentamicin formulations for two highly gentamicin-resistant mucoid and non-mucoid clinical strains of P. aeruginosa were significantly lower than the MICs (Table 1) of free gentamicin (1–2 versus 256–512 mg/L). Similarly, the MICs of the liposomal gentamicins for low or moderate gentamicin-resistant strains were at least one-half of the MICs of free gentamicin for the same organisms (P ≤ 0.05). Overall, no significant difference was observed in the activities of liposomal gentamicin formulations due to their lipid composition. Liposomes containing PBS or a combination of empty liposomes with free drug had no additive effect on gentamicin's antibacterial activity.6


View this table:
[in this window]
[in a new window]
 
Table 1. In vitro activities of free and liposomal gentamicin against P. aeruginosa isolated from cystic fibrosis patients

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The encapsulation efficiency of our liposomal formulations (4%–5%) is higher than the values reported by some investigators, such as Lutwyche et al.,7 who reported an encapsulation efficiency of <3% for a liposomal gentamicin composed of DPPC-CHOL (55:45, molar ratio). However, higher encapsulation efficiencies have been reported by other researchers.8 Liposomes have a low melting temperature (37.5°C) due to the lack of cholesterol and they begin to destabilize at 33°C. Therefore, we chose to incorporate cholesterol in our formulations in order to enhance the stability of our liposomes.

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 (MIC≥16 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.


    Acknowledgements
 
The authors thank Beverly Harper and Antoinetta Dell for their technical assistance. All clinical isolates of P. aeruginosa were kindly provided by the Department of Microbiology, Memorial Hospital, Sudbury, ON, Canada. This work was partly supported by a research grant from LURF (Laurentian University Research Funds).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Hubert, D. (2003). Cystic fibrosis in adults. Revue du Praticien 53, 158–62.[Medline]

2 . Hocquet, D., Bertrand, X., Kohler, T. et al. (2003). Genetic and phenotypic variations of a resistant Pseudomonas aeruginosa epidemic clone. Antimicrobial Agents and Chemotherapy 47, 1887–94.[Abstract/Free Full Text]

3 . Kallinteri, P., Antimisiaris, S. G., Karnabatidis, D. et al. (2002). Dexamethasone incorporating liposomes: an in vitro study of their applicability as a slow releasing delivery system of dexamethasone from covered metallic stents. Biomaterials 23, 4819–26.[CrossRef][ISI][Medline]

4 . Sentjurc, M., Vrhovnik, K. & Kristl, J. (1999). Liposomes as a topical delivery system: the role of size on transport studied by the EPR imaging method. Journal of Controlled Release 59, 87–97.[CrossRef][ISI][Medline]

5 . Omri, A., Suntres, Z. E. & Shek, P. N. (2002). Enhanced activity of liposomal polymyxin B against Pseudomonas aeruginosa in a rat model of lung infection. Biochemical Pharmacology 64, 1407–13.[CrossRef][ISI][Medline]

6 . Omri, A., Ravaoarinoro, M. & Poisson, M. (1995). Incorporation, release and in-vitro antibacterial activity of liposomal aminoglycosides against Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy 36, 631–9.[Abstract]

7 . Lutwyche, P., Cordeiro, C., Wiseman, D. J. et al. (1998). Intracellular delivery and antibacterial activity of gentamicin encapsulated in pH-sensitive liposomes. Antimicrobial Agents and Chemotherapy 42, 2511–20.[Abstract/Free Full Text]

8 . Beaulac, C., Sachetelli, S. & Lagacé, J. (1999). Aerosolization of low phase transition temperature liposomal tobramycin as a dry powder in an animal model of chronic pulmonary infection caused by Pseudomonas aeruginosa. Journal of Drug Targeting 7, 33–41.[ISI][Medline]

9 . Bernsdorff, C., Reszka, R. & Winter, R. (1999). Interaction of the anticancer agent Taxol (paclitaxel) with phospholipid bilayers. Journal of Biomedical Materials Research 46, 141–9.[ISI][Medline]

10 . Yasui, K., Fujioka, H. & Nakamura, Y. (1995). Controlled release by Ca2 + -sensitive recombinant human tumor necrosis factor-alpha liposomes. Chemical and Pharmaceutical Bulletin (Tokyo) 43, 508–11.





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
55/2/269    most recent
dkh518v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (2)
Disclaimer
Request Permissions
Google Scholar
Articles by Mugabe, C.
Articles by Omri, A.
PubMed
PubMed Citation
Articles by Mugabe, C.
Articles by Omri, A.