JAC

Journal of Antimicrobial Chemotherapy (2005) 55, 269271

doi:10.1093/jac/dkh518
Advance Access publication 8 December 2004

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; 2The University of Texas Health Center,
Department of Medicine, 11937 US Highway 271, Tyler, Texas 75708, USA

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-distearoylsn-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 378C in PBS and
at 378C 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 378C. 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

Materials and methods

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.

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

..........................................................................................................................................................................................................................................................................................................................................................................................................................

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

269
JAC vol.55 no.2 q The British Society for Antimicrobial Chemotherapy 2004; all rights reserved.

Downloaded from http://jac.oxfordjournals.org/ by guest on March 10, 2014

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

C. Mugabe et al.
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
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 g
for 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 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 (PA48912 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 Students t-test and
_ 0.05 was considered significant. For multiple comparisons
P<
within and between groups, ANOVA with the two-tailed Dunnetts
post-test analysis was used.

Results

Table 1. In vitro activities of free and liposomal gentamicin against P. aeruginosa isolated from cystic
fibrosis patients
MIC (mg/L)
non-mucoid strains
Gentamicin
formulations
Free
DMPC-CHOL
DPPC-CHOL
DSPC-CHOL

mucoid strains

PA-1

PA-48912 2

M-57192R

PA-48912 1

PA-48913

M-26250

16
8
4
4

32
8
4
4

512
2
8
8

32
1
0.5
2

256
1
1
2

16
8
4
1

a
Two-fold dilution series of free and liposomal gentamicin were prepared in triplicate in a broth medium and mixed with
100 mL of P. aeruginosa (1.5107 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. Two
control strains, P. aeruginosa (ATCC 27853) and S. aureus (ATCC 29213), were used to validate this method. Two separate experiments were performed and no significant differences in the MIC values were observed.

270

Downloaded from http://jac.oxfordjournals.org/ by guest on March 10, 2014

Figure 1. Gentamicin retention in liposomes of various lipid compositions

in human plasma. Liposome-encapsulated gentamicin composed of DMPCCHOL (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 (centrifugation at 18300 g for 15 min at 48C) and gentamicin concentrations in the
supernatants were determined by agar diffusion microbiological assay.
Results are meansS.E.M . of three separate experiments. All formulations
retained a comparable amount of gentamicin at the end of a 48 h assay
period.

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 formuladependent and varied significantly (P < 0.001) from 26.7 1.3 to
34.5 1.5 mg/mmol.
All liposomal preparations retained >70% of the initially
encapsulated gentamicin up to 48 h in PBS. Generally, however,
liposomes incubated at 48C retained more antibiotic than those
incubated at 378C. For instance, the liposomes composed of
DPPC-CHOL (DPPCcholesterol) retained significantly more
antibiotic at 48C than those stored at 378C (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 DMPCCHOL at 48C (89.3% 1.3% versus 77.3% 1.1%, P < 0.0003)
in 48 h. Likewise, the liposomes composed of DPPC-CHOL

Liposomal gentamicin

Discussion
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 (37.58C) due to the lack
of cholesterol and they begin to destabilize at 338C. 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 48C than liposomes composed of DSPCCHOL 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 antitumour 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 378C, they

performed well when compared with liposomes composed of

egg phosphatidylcholine.10
All clinical strains of P. aeruginosa used in this study are
_ 16 mg/L).
considered resistant (NCCLS) to gentamicin (MIC>
Our formulations, however, significantly enhanced the susceptibilities of these organisms to gentamicin, from highly
_ 8 mg/L) or susceptible
resistant to either intermediate (MIC<
_ 4 mg/L) to this antimicrobial agent. Liposomes may pro(MIC<
tect 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
1. Hubert, D. (2003). Cystic fibrosis in adults. Revue du Praticien
53, 15862.
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,
188794.
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.
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.
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,
140713.
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, 6319.
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.
8. Beaulac, C., Sachetelli, S. & Lagace, 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.
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.
10. Yasui, K., Fujioka, H. & Nakamura, Y. (1995). Controlled
release by Ca2 + -sensitive recombinant human tumor necrosis factoralpha liposomes. Chemical and Pharmaceutical Bulletin (Tokyo) 43,
508 11.

271

Downloaded from http://jac.oxfordjournals.org/ by guest on March 10, 2014

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 378C did not indicate any significant difference in
the lipid compositions.
To mimic physiological conditions, we determined the drugrelease kinetics of liposomal gentamicin incubated in normal
human pooled plasma at 378C (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 378C 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 378C in PBS, compared with
64.1% 4.3% (P < 0.0001) of the same formulation in plasma.
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 256512 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
_ 0.05). Overall, no
free gentamicin for the same organisms (P <
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 gentamicins antibacterial
activity.6