Liposome-mediated gentamicin delivery: development and activityagainst resistant strains of Pseudomonas aeruginosa isolated

from cystic fibrosis patients

Clement Mugabe1, Ali O. Azghani2 and Abdelwahab Omri1*

1The Novel Drug&Vaccine Delivery Systems Facility, Department of Chemistry and Biochemistry, Laurentian

University, 935 Ramsey Lake Rd, Sudbury, Ontario, P3E 2C6, Canada; 2The 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

Objectives: Chronic pulmonary infection by Pseudomonas aeruginosa in cystic fibrosis patients isvirtually impossible to eradicate by means of existing free antibiotics. We sought to assess the anti-bacterial activities of liposomal gentamicin against clinical isolates of P. aeruginosa.

Methods: Gentamicin was encapsulated into liposomes with different lipid compositions (1,2-dimyris-toyl-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 vitrostability of liposome-encapsulated gentamicin was studied over a 48 h period at 4 and 378C in PBS andat 378C in pooled plasma. The MICs of free and liposomal gentamicin for clinical isolates of P. aerugi-nosa were assessed by broth dilution.

Results: The encapsulation efficiency of all liposomal preparations was 4%–5.18% of the initial amountof the drug in solution. The liposomes retained 60%–70% of the encapsulated gentamicin for 48 hwhen they were incubated in normal human pooled plasma or PBS at 4 or 378C. The MICs of liposomalgentamicin for all clinical isolates of P. aeruginosa were lower than the MICs of free gentamicin. Impor-tantly, liposomal gentamicin altered the susceptibilities of these clinical isolates from gentamicinresistant to either intermediate or susceptible.

Conclusions: Taken together, these data indicate that liposomal gentamicin formulations could bemore effective than the free drug in controlling pulmonary infections due to P. aeruginosa.

Keywords: antibiotic delivery, lung infection, stability

Introduction

Pseudomonas aeruginosa is a major cause of nosocomial infec-tions and accounts for � 95% of deaths in the cystic fibrosispopulation.1 A major reason for its prominence as a pathogen isits high intrinsic resistance to most available antibiotics.2 Adrug-delivery system that could reduce antibiotic toxicity whileincreasing the therapeutic indices is of great interest, andliposome-encapsulated antimicrobial agents can provide thesebenefits.

The present study was undertaken to evaluate encapsulationefficiency, in vitro stability and antibacterial activity of ournewly developed liposomal gentamicin formulations againstresistant strains of P. aeruginosa.

Materials and methods

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 gen-

tamicin 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

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*Corresponding author. Tel: +1-705-675-1151 ext. 2190; Fax: +1-705-675-4844; E-mail: aomri@nickel.laurentian.ca..........................................................................................................................................................................................................................................................................................................................................................................................................................

Journal of Antimicrobial Chemotherapy (2005) 55, 269–271

doi:10.1093/jac/dkh518

Advance Access publication 8 December 2004JAC

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the content of total lipid. The concentration of gentamicin incorpor-

ated 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

378C 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 gfor 15 min at 48C) 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 percen-

tage 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 con-

trol. 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

The average diameter of liposome-encapsulated gentamicin withdifferent lipid compositions varied from 408 ± 28 to 418 ± 21 nm,with no significant difference between various liposomal prep-arations. The polydispersity index [the measurement ofhomogeneity of dispersion, ranging from 0.0 (homogeneous) to1.0 (heterogeneous)] for the size distribution of liposomal formu-lations ranged from 0.59 ± 0.009 to 0.74 ± 0.007. Encapsulationefficiencies of gentamicin in all three types of liposomes were4%–5.18% of the initial amount of the drug in solution. Theloading capacity of gentamicin, however, was formula-dependent and varied significantly (P < 0.001) from 26.7 ± 1.3 to34.5 ± 1.5mg/mmol.

All liposomal preparations retained >70% of the initiallyencapsulated gentamicin up to 48 h in PBS. Generally, however,liposomes incubated at 48C retained more antibiotic than thoseincubated at 378C. For instance, the liposomes composed ofDPPC-CHOL (DPPC–cholesterol) retained significantly moreantibiotic at 48C than those stored at 378C (89.3% ± 1.3% versus80.6% ± 1.2%, P < 0.0001) at the end of a 48 h experimentalperiod. We also compared the drug release profile of differentliposomal formulations and found that the liposomes composedof DPPC-CHOL retained more antibiotic than that of DMPC-CHOL at 48C (89.3% ± 1.3% versus 77.3% ± 1.1%, P < 0.0003)in 48 h. Likewise, the liposomes composed of DPPC-CHOL

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 378C in normal human pooled plasma with mild

agitation. At the times indicated, plasma samples were removed (centrifu-

gation at 18300 g for 15 min at 48C) 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.

Table 1. In vitro activities of free and liposomal gentamicin against P. aeruginosa isolated from cystic

fibrosis patients

MIC (mg/L)

non-mucoid strains mucoid strains

Gentamicinformulations PA-1 PA-48912–2 M-57192R PA-48912–1 PA-48913 M-26250

Free 16 32 512 32 256 16DMPC-CHOL 8 8 2 1 1 8DPPC-CHOL 4 4 8 0.5 1 4DSPC-CHOL 4 4 8 2 2 1

a Two-fold dilution series of free and liposomal gentamicin were prepared in triplicate in a broth medium and mixed with100mL of P. aeruginosa (1.5�107 cfu/mL). The test tubes were then incubated at 378C for 24 h in a shaking water bath.The MICs were determined as the lowest concentration of the antibiotics that inhibited visible bacterial growth. Twocontrol strains, P. aeruginosa (ATCC 27853) and S. aureus (ATCC 29213), were used to validate this method. Two sepa-rate experiments were performed and no significant differences in the MIC values were observed.

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retained more antibiotic than that of DSPC-CHOL(89.3% ± 1.3% versus 84.1% ± 1.2%, P < 0.005) at the end of a48 h experimental period. However, the drug-release dataobtained at 378C did not indicate any significant difference inthe lipid compositions.

To mimic physiological conditions, we determined the drug-release kinetics of liposomal gentamicin incubated in normalhuman pooled plasma at 378C (Figure 1). All liposomal formu-lations released � 40% of the encapsulated drug in 48 h.Although we did not detect a significant difference in the releasekinetics of gentamicin in plasma in relation to different liposo-mal formulations, we found that the liposomes incubated in PBSat 378C retained significantly more antibiotic than when theywere incubated in plasma over a period of 48 h. For instance, theliposomes composed of DPPC-CHOL retained 80.6% ± 1.2% ofinitially encapsulated drug at 378C in PBS, compared with64.1% ± 4.3% (P < 0.0001) of the same formulation in plasma.

The MICs of all three liposomal gentamicin formulations fortwo highly gentamicin-resistant mucoid and non-mucoid clinicalstrains of P. aeruginosa were significantly lower than the MICs(Table 1) of free gentamicin (1–2 versus 256–512 mg/L). Simi-larly, the MICs of the liposomal gentamicins for low or moderategentamicin-resistant strains were at least one-half of the MICs offree gentamicin for the same organisms (P <_ 0.05). Overall, nosignificant difference was observed in the activities of liposomalgentamicin formulations due to their lipid composition. Lipo-somes containing PBS or a combination of empty liposomes withfree drug had no additive effect on gentamicin’s antibacterialactivity.6

Discussion

The encapsulation efficiency of our liposomal formulations(4%–5%) is higher than the values reported by some investi-gators, such as Lutwyche et al.,7 who reported an encapsulationefficiency of <3% for a liposomal gentamicin composed ofDPPC-CHOL (55:45, molar ratio). However, higher encapsula-tion efficiencies have been reported by other researchers.8 Lipo-somes have a low melting temperature (37.58C) due to the lackof cholesterol and they begin to destabilize at 338C. Therefore,we chose to incorporate cholesterol in our formulations in orderto enhance the stability of our liposomes.

Our drug-release data reported here clearly indicate a signifi-cant improvement in gentamicin retention by our formulations,regardless of the temperature. The contributing factors includeour choice of lipids, the ratio of lipid to cholesterol (2:1, molarratio) and the chemical nature of the antibiotic, gentamicin. Wefound that liposomes composed of DPPC-CHOL retained moreantibiotic in PBS at 48C than liposomes composed of DSPC-CHOL or DMPC-CHOL. Although at present we do not knowwhy the DPPC-CHOL formula increases the stability of theseliposomes in PBS, other studies echo similar findings. It hasbeen reported that the DPPC-encapsulated paclitaxel, an anti-tumour agent, is more stable in PBS than the liposomes com-posed of DMPC or DSPC.9 These authors argued that paclitaxelincorporation increases the phase-transition temperature of thelipid vesicles and that this broadening effect was greatest forDPPC, hence leading to a more stable compound. Although ourformulations incubated in normal human pooled plasma released20% more antibiotic than those incubated in PBS at 378C, they

performed well when compared with liposomes composed ofegg phosphatidylcholine.10

All clinical strains of P. aeruginosa used in this study areconsidered resistant (NCCLS) to gentamicin (MIC>_16 mg/L).Our formulations, however, significantly enhanced the suscep-tibilities of these organisms to gentamicin, from highlyresistant to either intermediate (MIC<_8 mg/L) or susceptible(MIC<_4 mg/L) to this antimicrobial agent. Liposomes may pro-tect the encapsulated drug from the action of bacterial enzymesas well as facilitating its diffusion across the bacterial envelope.To the best of our knowledge, however, this is the first report onliposomal formulations that enhance gentamicin antibacterialactivity against gentamicin-resistant clinical strains of P. aerugi-nosa. We are investigating the mechanism by which theseformulations enhance gentamicin activity against the resistantstrains of P. aeruginosa.

Acknowledgements

The authors thank Beverly Harper and Antoinetta Dell for theirtechnical assistance. All clinical isolates of P. aeruginosa werekindly provided by the Department of Microbiology, MemorialHospital, Sudbury, ON, Canada. This work was partly supportedby a research grant from LURF (Laurentian University ResearchFunds).

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