Journal of Antimicrobial Chemotherapy (2008) 62, 1291 –1297

doi:10.1093/jac/dkn422
Advance Access publication 18 October 2008

Co-encapsulation of gallium with gentamicin in liposomes enhances

antimicrobial activity of gentamicin against Pseudomonas aeruginosa

M. Halwani1, B. Yebio1, Z. E. Suntres1,2, M. Alipour1, A. O. Azghani3 and A. Omri1*

1

Downloaded from https://academic.oup.com/jac/article-abstract/62/6/1291/773926 by guest on 28 November 2019

The Novel Drug and Vaccine Delivery Systems Facility, Department of Chemistry and Biochemistry, Laurentian
University, Sudbury, Ontario, Canada P3E 2C6; 2Medical Sciences Division, Northern Ontario School of
Medicine, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario, Canada P7B 5E1; 3Department of
Biology, University of Texas at Tyler, 3900 University Boulevard, Tyler, TX 75799, USA

Received 18 May 2008; returned 27 July 2008; revised 12 September 2008; accepted 14 September 2008

Objectives: The aim of this study was to enhance the antimicrobial efficacy of a liposomal gentamicin
formulation with gallium metal (Lipo-Ga-GEN) against clinical isolates of Pseudomonas aeruginosa.
Methods: Sputum isolates of P. aeruginosa from cystic fibrosis patients were used to determine the
MIC and MBC of Lipo-Ga-GEN. P. aeruginosa biofilms were formed and used to compare the minimum
biofilm eradication concentration of the conventional drugs with that of Lipo-Ga-GEN. Quorum sensing
(QS) molecule reduction of P. aeruginosa was determined by monitoring N-acyl homoserine lactone
production using Agrobacterium tumefaciens reporter strain (A136). Viability of the cultured human
lung epithelial cells (A549) was determined by Trypan Blue assay in order to assess Ga toxicity.
Results: MIC and MBC values indicated that gentamicin was more effective against a highly resistant
strain of P. aeruginosa (PA-48913) when delivered as a Lipo-Ga-GEN formulation (256 mg/L free genta-
micin versus 2 mg/L Lipo-Ga-GEN). Lipo-Ga-GEN was the only formulation that completely eradicated
biofilms and blocked QS molecules at a very low concentration (0.94 mg/L gentamicin). The decrease
in cell viability was less in A549 cells exposed to Lipo-Ga, suggesting that encapsulated Ga is safer.
Conclusions: The results clearly indicate that the Lipo-Ga-GEN formulation is more effective than genta-
micin alone in eradicating antibiotic-resistant P. aeruginosa isolates growing in a planktonic or biofilm
community.

Keywords: antibiotics, aminoglycosides, biofilms, quorum sensing, toxicity

Introduction membrane permeability, an effect attributed to the alterations in

exopolysaccharide and alginate biofilm production,8,9 which is
Cystic fibrosis (CF) is a chronic disease with no cure available controlled by bacterial communication through quorum sensing
at the present time. However, efforts in the development of gene (QS) signal molecules.10,11
therapy to restore the CF transmembrane conductance regulator Liposomes, as a drug carrier system, have the capacity to opti-
(CFTR) function might improve outcomes.1 Equally important mize antibiotic therapy by reducing antibiotic toxicity, improving
is the improvement of existing therapeutics to prevent emerging drug uptake and enhancing bactericidal efficacy through fusion
antibiotic-resistant bacteria common to CF.2 CFTR chloride with the bacterial membrane.12,13 Recently, gallium has emerged
channel deficiency or absence results in an abnormal epithelia as an effective inhibitory agent against P. aeruginosa growth and
lining fluid suitable for microbial colonization and growth.3 biofilm formation. Disruption of iron metabolism increases the
Aminoglycoside antibiotics such as gentamicin are not only vulnerability of most infecting bacteria because iron is essential
highly effective against Pseudomonas aeruginosa but can also for growth and the functioning of key enzymes, such as those
suppress the expression of some of the nonsense mutations of involved in protein and DNA synthesis, electron transport and
the CFTR, hence allowing it to function normally.4,5 However, oxidative stress.14,15 It is recognized that Ga, due to its chemical
gentamicin use is restricted due to the emerging resistant similarities to Fe, can substitute for Fe in many biological
strains.6,7 Bacteria can become resistant by changing their outer systems and inhibits Fe-dependent processes.16,17

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

*Corresponding author. Tel: þ1-705-675-1151, ext. 2190; Fax: þ1-705-675-4844; E-mail: aomri@laurentian.ca
.....................................................................................................................................................................................................................................................................................................................................................................................................................................

1291
# The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
For Permissions, please e-mail: journals.permissions@oxfordjournals.org
 Halwani et al.
This study was carried out in order to develop a liposomal
formulation that could co-deliver gentamicin and Ga in an effort
to improve gentamicin efficacy. Although liposomal delivery of
gentamicin is known to increase its antibacterial efficacy, its
co-delivery with Ga would further improve its effects by reducing
biofilm formation. In this study, the properties of the new formu-
lation, Lipo-Ga-GEN, in terms of its stability, antibacterial
activity, prevention of P. aeruginosa N-acyl homoserine lactone
(AHL) production (a QS molecule) and biofilm formation, and
reduction of Ga cell toxicity in vitro were investigated.
Materials and methods
Chemicals and media
Liposomes were prepared from 1,2-dipalmitoyl-sn-glycero-3-
phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-[phospho-
rac-glycerol] (DMPG) phospholipids (Northern Lipids, Vancouver,
BC, Canada). Cholesterol, Ga (III) nitrate and chemical reagent X-gal
were purchased from Sigma–Aldrich (Oakville, ON, Canada). The
citrated human pooled plasma was obtained from Precision- Biologic
(Dartmouth, NS, Canada). All other products were obtained from
Fisher Scientific (Ottawa, ON, Canada).
Cell culture
For the toxicity study, the alveolar type II-like epithelial cells iso-
lated from the human lung carcinoma (A549) cell line were used
(ATCC CCL-185; ATCC, Manassas, VA, USA). Cells were grown
in Dulbecco’s modified Eagle’s medium supplemented with 10%
heat-inactivated fetal bovine serum. The cells were maintained at
378C in 5% CO2 and were utilized at around 85% confluence.
Bacterial strains
Clinical isolates of P. aeruginosa (PA-48912-2, PA-48913 and
PA-48912-1) were obtained from CF sputum (Sudbury Regional
Hospital, ON, Canada). Laboratory strains of Staphylococcus aureus
ATCC 29213 were used as quality control. All strains were stored in
Mueller –Hinton broth at 2808C, supplemented with 10% glycerol,
and subcultured for 18 h in Mueller –Hinton broth prior to experi-
ments. Agrobacterium tumefaciens strains A136 (Ti2) ( pCF218)
( pCF372) and KYC6 were used in the QS experiments and cultured
in Luria–Bertani (LB) broth at 308C, supplemented with spectino-
mycin (50 mg/L), tetracycline (4.5 mg/L) and 15% glycerol for
storing at 2808C. Routine QS experiments were carried out by sub-
culturing strains in LB broth as mentioned earlier in the absence of
the antibiotics.
Lipo-Ga-GEN formulation preparation
The dehydration– rehydration technique was used to prepare multi-
lamellar vesicles containing Ga with entrapped gentamicin. Briefly,
DPPC and negatively charged DMPG (molar ratio 10:1) were
dissolved in chloroform. The lipid film was then rehydrated with
distilled water containing sucrose (1:1, w/v) and 500 mM dissolved
Ga (III) nitrate. The solution was sonicated for 5 min prior to the
addition of gentamicin (8 mg/mL) and, thereafter, sonicated for an
additional 5 min and lyophilized as reported elsewhere.18 To rehy-
drate, sterile water (10% of final volume before lyophilization) was
added, and the mixtures were incubated at 508C for 30 min. This
step was repeated once more with PBS. Additional PBS was then
1292
added to form the original volume and incubated as described
earlier. The rehydrated Lipo-Ga-GEN vesicles were centrifuged
(100 000 g for 20 min at 48C in a Beckman L8-M Ultracentrifuge)
and washed twice with PBS to remove the unencapsulated Ga and
gentamicin. The Submicron Nicomp particle sizer (Model 270,
Nicomp, Santa Barbara, CA, USA) was used to measure the dia-
meter of Lipo-Ga-GEN vesicles, as described previously.12
Gentamicin encapsulation efficiency (EE) within the
Lipo-Ga-GEN formulation
An agar diffusion method was used to determine the amount of
Downloaded from https://academic.oup.com/jac/article-abstract/62/6/1291/773926 by guest on 28 November 2019
encapsulated gentamicin in Lipo-Ga-GEN. Briefly, standard curves
for diluted gentamicin as well as samples of Lipo-Ga-GEN were
prepared. Liposomal samples were disrupted by 0.1% Triton X-100
for 30 min at 378C and then inoculated into the holes of the agar
plates containing S. aureus ATCC 29213 in duplicate, as well as
20 mM Ga as a control. Plates were incubated at 378C for 24 h, and
the inhibition zones were then measured. EE was calculated as
follows:
concentration of gentamicinreleased
EE ¼ 100
concentration of gentamicininitial
Ga quantification within the Lipo-Ga-GEN formulation
The Ga content in the Lipo-Ga-GEN formulation was measured by
graphite furnace atomic absorption spectroscopy (GFAAS).
Lipo-Ga-GEN samples were lyophilized, weighed and then trans-
ferred into Teflon tubes. A total of 1 mL of H2O2 (30%, w/w) and
4 mL of HNO3 (15 N) was added, and the samples were digested
overnight at 258C to release the liposomal content. Samples were
then subjected to heated sand bath digestion at 135–1408C for 4 h
to complete the release of any trace amount of Ga, and the volumes
were then adjusted to 25 mL with distilled water. Samples were ana-
lysed by GFAAS (Perkin-Elmer 5000).
Gentamicin stability within the Lipo-Ga-GEN formulation
Gentamicin leakage from Lipo-Ga-GEN was further measured after
exposure to bronchoalveolar lavage (BAL) fluid (obtained from rat’s
lung according to an approved procedure from the Laurentian
University Animal Care Committee18,19), pooled normal human
plasma and PBS at intervals of 0.5, 1, 3, 6, 24 and 48 h. Samples
were centrifuged (100 000 g, 20 min, 48C) and gentamicin concen-
trations in the supernatants were measured by agar diffusion methods
as described previously.12
Determination of MICs and MBCs
A broth dilution method was used to determine the MICs of genta-
micin.20 Briefly, strains of S. aureus and P. aeruginosa were exposed
to serial dilutions of free gentamicin or Lipo-Ga-GEN. The contri-
bution of Ga to the MICs was assessed by exposing the same bac-
terial strains to different concentrations of Ga added in the forms of
free Ga, Ga-GEN, Lipo-Ga and Lipo-Ga-GEN, with a starting con-
centration of 42 mM. Drug-free bacterial cultures and broth medium
alone were used as positive and negative controls, respectively. The
sub-MIC, MIC and two times the MIC were plated on agar and incu-
bated for a further 24 h at 378C to determine the MBCs.
 Liposomal-gallium-gentamicin formulation
Lipo-Ga-GEN bactericidal activity against P. aeruginosa
biofilms [minimum biofilm eradication concentration
(MBEC)]
Biofilms of P. aeruginosa strains (PA-48912-2 and PA-48912-1)
were produced by using Calgary biofilm plates (Innovotech,
Edmonton, AB, Canada), in order to assess the MBEC. The biofilms
were established on the plate lid pegs soaked in P. aeruginosa in
Mueller –Hinton broth for 72 h at 378C (fresh broth supplemented
every 24 h). On day 3, the formed biofilm pegs were transferred to
microcentrifuge tubes after removal by a sterile forcep into 1 mL of
PBS and sonicated to retrieve biofilm bacteria. Biofilms were
exposed to different dilutions of free Ga, Lipo-Ga, free gentamicin,
Lipo-GEN, Ga-GEN and Lipo-Ga-GEN for 24 h at 378C. Aliquots
of 100 mL of each sample were then plated on Mueller –Hinton agar
and incubated for 24 h at 378C. Finally, cfu/mL were counted to
determine the MBEC.
QS reduction assay
The QS reduction assay was performed by monitoring the production
of the AHL molecule, the main QS molecule that is produced by
P. aeruginosa after exposure of the bacterium to the Lipo-Ga-GEN
formulation. Samples of P. aeruginosa strain PA-48913 cultured for
18 h were standardized to a turbidity equivalent to that of a 1.0
McFarland standard and then treated with various concentrations of
free Ga, Lipo-Ga, free gentamicin, Lipo-GEN, Ga-GEN or
Lipo-Ga-GEN for 1 h at 378C. Samples of PBS-treated P. aeruginosa
were used as positive controls. The samples were then centrifuged for
15 min (18 000 g) at 48C; the pellets were discarded and the super-
natants were assayed for AHL production by the following procedure:
mixed sterilized LB agar with A. tumefaciens A136 (Ti2) (pCF218)
(pCF372) was solidified in a glass plate with 1 mL of X-gal reagent
(20 mg/mL in dimethylformamide). The supernatants were poured
into wells of the plate. After incubation for 24 h at 378C, the edge of
the holes was inspected for a blue pigmentation, an AHL indicator.
Samples of A136 supernatant served as negative controls as the over-
exposure of A136 to itself inhibits AHL production. To rule out the
bactericidal effects on AHL production, the cfu/mL of cultures of
untreated P. aeruginosa PA-48913 incubated at 378C for 24 h was
compared with that of the samples treated with the lowest concen-
tration of Lipo-Ga-GEN that prevented AHL production.
Lipo-Ga toxicity study
A549 cells at a density of 5  105 cells/dish were allowed to adhere
and grow overnight. The medium was replaced with 2 mL of
medium containing 7.5, 15, 30 or 60 mM Ga in either free form or
within Lipo-Ga formulations. Controls included untreated cells
containing medium alone, cells treated with H2O2 as a positive
control, empty liposomes (weight equal to 60 mM Lipo-Ga-GEN)
and different concentrations of free gentamicin (10–1000 mg/L). All
dishes were incubated for 24 h in 5% CO2 at 378C. The medium was
removed and the cells were washed with Dulbecco’s PBS three
times. The viable cells were detached by trypsin/EDTA treatment
for 30 s, and samples were centrifuged for 4 min at 48C. The pellets
were resuspended in 2 mL of fresh medium and viability was
measured by Trypan Blue assay.
Data analysis
Data are presented as means + SEM of three independent experi-
ments. Comparisons between individual columns and groups were
made by paired Student’s t-test, and *P  0.05, **P  0.01 or
1293
***P  0.001 was considered significant. For multiple comparisons
within and between groups, we used ANOVA with one- and
two-way analysis. All analyses were performed using GraphPad
Prism, version 5.0.
Results
Lipo-Ga-GEN stability
Preparation of Lipo-Ga-GEN liposomes produced multilamellar
vesicles of 337 + 35 nm. The EE for gentamicin in the Lipo-Ga-
GEN formulation was 0.34 mg/mL. The concentration of Ga incor-
porated into the Lipo-Ga-GEN formulation was 19 + 0.15 mM, as
Downloaded from https://academic.oup.com/jac/article-abstract/62/6/1291/773926 by guest on 28 November 2019
determined by atomic absorption analysis. Lipo-Ga-GEN retained
.92% of gentamicin in PBS at 48C and in BAL at 378C for 48 h.
However, gentamicin was significantly released (43%) from the
formulation in plasma for 48 h at 378C (Figure 1).
MIC and MBC determination
The MICs of Lipo-Ga-GEN for all P. aeruginosa strains exam-
ined were significantly lower than those of free gentamicin and
the Ga-GEN combination. As depicted in Table 1, the MIC of
free gentamicin for P. aeruginosa PA-48912-1 was 64 mg/L
compared with 32 mg/L Ga-GEN and 4 mg/L Lipo-Ga-GEN.
The latter formulation was bactericidal at 8 mg/L, as indicated by
the MBC values in Table 2. The difference between the MICs
and MBCs of Lipo-Ga-GEN and free gentamicin for the
antibiotic-resistant P. aeruginosa PA-48913 strain was remark-
able (2 to 4 versus 256 to .512 mg/L gentamicin). MICs of free
gentamicin for S. aureus ATCC 29213 were comparable with
CLSI values (data not shown).
Lipo-Ga-GEN bactericidal activity against P. aeruginosa
biofilm
The Lipo-Ga-GEN formulation activity against the biofilm
community of P. aeruginosa PA-48912-1 and PA-48912-2 was
Gentamicin stability within Lipo-Ga-GEN formulation
100
90
Retention (%)
80
** ** **
** **
70
PBS (4°C) **
60
BAL (37°C)
Plasma (37°C)
50
0 0.5 1 3 6 24 48
Time (h)
Figure 1. Lipo-Ga-GEN formulation stability in PBS at 48C (open squares),
in BAL at 378C (filled circles) and in plasma at 378C (filled triangles). Data
represent the means + SEM of three independent experiments in duplicate.
P values relate to the comparison of the BAL and plasma conditions with
the control PBS condition; **P  0.01, significant.

Table 1. MIC comparison of the drug formulations
Bacterial strain Ga (mM) GEN
PA-48912-1 .42 64
PA-48913 .42 256
MICs of free gentamicin, free gallium, a combination of free gallium with free gentamicin, Lipo-Ga; Lipo-GEN and Lipo-Ga-GEN for resistant isolates of P.
aeruginosa (PA).
a
Ga values (2.6 and 10.5 mM, respectively).
b
Ga values (0.3 and 0.16 mM, respectively).
Table 2. MBC comparison of the drug formulations
Bacterial strain Ga (mM) GEN
PA-48912-1 .42 128
PA-48913 .42 .512
MBCs of free gentamicin, free gallium, a combination of free gallium with free gentamicin, Lipo-Ga; Lipo-GEN and Lipo-Ga-GEN for resistant isolates of P.
aeruginosa (PA).
a
Ga values (5.25 and 21 mM, respectively).
b
Ga values (0.6 and 0.3 mM, respectively).
determined to mimic the in vivo condition of the CF lungs. As
shown in Figure 2, Lipo-Ga-GEN was the only formulation that
eliminated these strains in a biofilm format at 8 mg/L, while
Lipo-GEN was significantly better than the free gentamicin at
reducing biofilm. It was also observed that the liposomal
formula improved gentamicin bactericidal activity.
QS reduction assay
The Lipo-Ga-GEN formulation interrupted bacterial QS
signalling elements at a concentration lower than its MIC for
P. aeruginosa. Lipo-Ga-GEN eliminated AHL production by the
P. aeruginosa PA-48913 strain at 0.94 mg/L gentamicin and
0.08 mM Ga. Although Lipo-GEN slightly inhibited the AHL
production, other Ga- and/or gentamicin-containing formulations
were ineffective.
Toxicological effect of Lipo-Ga formulation
In order to rule out that gentamicin is toxic to lung epithelial
cells, A549 cells were incubated with different concentrations of
this antibiotic. The viability of the A549 cell line was studied
with free Ga and Lipo-Ga in the absence of gentamicin. The
exposure of A549 cells to different concentrations of free Ga
resulted in a concentration-dependent reduction in cell viability.
In contrast, Lipo-Ga was not as toxic. For example, 74.2 + 6.8%
of the cells were viable after exposure to 15 mM Lipo-Ga,
whereas Ga at a similar concentration reduced the monolayer
viability significantly (47.8 + 3.0%, P  0.001) (Figure 3). The
exposure of cells to empty liposomal formulations composed of
DPPC and negatively charged DMPG did not alter cell viability,
and data were comparable with those of DPPC and neutral
cholesterol (data not shown).
Halwani et al.
MIC of gentamicin (mg/L)
Ga-GENa Lipo-Ga (mM) Lipo-GEN Lipo-Ga-GENb
32 .42 16 4
128 .42 8 2
Downloaded from https://academic.oup.com/jac/article-abstract/62/6/1291/773926 by guest on 28 November 2019
MBC of gentamicin (mg/L)
Ga-GENa Lipo-Ga (mM) Lipo-GEN Lipo-Ga-GENb
64 .42 32 8
256 .42 16 4
Discussion
The lack of availability of newer antibiotics and the emergence
of pathogens resistant to the conventional antibiotics have
shifted research to the optimization of the existing drugs. In
addition, several antibiotics, including gentamicin, are ineffec-
tive in penetrating bacterial biofilm communities within the
tissues or on medical devices.21,22 Bacterial biofilms are respon-
sible for many persistent and chronic bacterial infections due to
their inherent resistance to antimicrobial agents.23
The liposomal antibiotic delivery system appears to be an
attractive strategy for penetrating biofilm barriers, targeting the
infection sites and improving the drug’s residence time and
uptake by bacteria.24 – 26 Furthermore, this biologically inert
system reduces drug-associated adverse effects.27,28 In this study,
an attempt was made to resolve bacterial resistance by not only
using antibiotics to kill the bacteria but also to prevent their
ability to produce biofilm. Accordingly, the liposomal delivery
system was used to co-deliver gentamicin and Ga, a metal
known to inhibit biofilm formation by interrupting Fe metab-
olism, important in the development of biofilm production.16,29
Recent studies have shown that liposomes can effectively pene-
trate biofilms.30
For this study, the negatively charged liposomal formulation
was developed from DPPC and DMPG phospholipids in a molar
ratio of 10:1 in an effort to co-encapsulate the two positively
charged molecules of gentamicin and Ga within the liposome
vesicles.31 This formulation demonstrated stability (.92% to
93% for gentamicin) and proximity of 70% of Ga EE, which
was maintained at different environments for a prolonged
period of time. However, further studies are required to confirm
the size of vesicles in relation to Ga. Plasma and BAL com-
ponents such as lipoproteins, albumin, immunoglobulins and
1294
 Liposomal-gallium-gentamicin formulation

Lipo-Ga-GEN Bactericidal activity against P. aeruginosa biofilm effective at reducing the rate of the P. aeruginosa antibiotic
10 resistance development phenomenon. This finding is consistent
PA-48912-1 with the results reported by Peeters et al.,36 who showed that Ga
8 PA-48912-2 had a limited effect on Burkholderia cepacia strains in the pre-
sence or absence of iron; impermeability of those strains to
log cfu/mL

6 gentamicin was due to changes in LPS structure.36,37 However,

*** *** *** ***
we observed a slight enhancement of gentamicin bacterial
***
4 killing when incorporated within liposomes due to their fusible
***
property with bacterial membranes.12 On the other hand,
2 Lipo-Ga-GEN inhibited the growth of gentamicin-resistant
*** *** strains of P. aeruginosa and also enhanced Ga uptake, which
0
clearly supports our hypothesis of using two antibacterial
agents.

Downloaded from https://academic.oup.com/jac/article-abstract/62/6/1291/773926 by guest on 28 November 2019

l

EN

EN

EN

EN
ro

-G
Furthermore, in the in vitro model of P. aeruginosa biofilm,
nt

-G

G
ee

po
Co

a-

a-
Fr

ee

po the biofilm bacteria were aggressively resistant to free genta-

Li
G

-G
Fr

Li

po micin, Ga and the combination of Ga-GEN without any eradica-

Li
MBEC
Figure 2. MBEC assay: antibacterial effect on PA-48912-1 (white bars) and
PA-48912-2 (black bars). Lipo-Ga-GEN (0.6 mM and 8 mg/L) was the only
formulation that inhibited the bacterial growth completely at 8 mg/L gentamicin
compared with free gentamicin (8 mg/L), free Ga (0.6 mM), Ga-GEN (0.6 mM
and 8 mg/L), Lipo-Ga (0.6 mM) and Lipo-GEN (8 mg/L). Data were obtained
from three independent experiments and are shown as means + SEM. P values
compare each formulation with the control; ***P  0.001.
phospholipases are critical factors that destabilize liposomes
dependent on lipid composition or the electrostatic charge.32,33
Destabilization of liposomes results in the perturbation of lipo-
somal structural integrity and permeability properties with
leakage of the entrapped agents. It has been shown that lipo-
somes containing only one class of negatively charged phospho-
lipids bound a great amount of protein and were very unstable;
however, those liposomes also containing phosphatidylcholine
(DPPC) bound less protein and were more stable.34 The addition
of sucrose as a lyoprotectant in the freeze-drying process might
also contribute to the stability of the liposome particles.35
Despite the antimicrobial activity of Lipo-Ga-GEN, free gen-
tamicin, free Ga and the combination Ga-GEN were not as
Toxicity of Lipo-Ga formulation
100
**
***
80
***
Cell viability (%)
tion emergence; also, a slight enhancement of the gentamicin
bactericidal activity was observed when incorporated within the
liposomes, while Lipo-Ga-GEN was the only formulation that
enabled complete eradication of the biofilm bacteria. The data of
this study confirmed the findings of other investigators on the
biofilm inhibitory effects of Ga.16 It might be possible that the
liposomal vesicles played an important role in maintaining a
continuous and longer contact between the drugs and the
bacterial biofilm, which might accelerate biofilm penetration as
suggested by other investigators as well.30
In an effort to understand the mechanism(s) of action of the
formulation on biofilm population, we addressed the question of
whether Lipo-Ga-GEN suppresses QS molecules such as AHL,
as it is one of the communication tools for biofilm for-
mation.29,38 It was shown that the liposomal formulation
suppressed AHL production without killing the bacteria. The
detailed molecular mechanism(s) involved in QS inhibition by
the liposomal formulation is currently under investigation in our
laboratory.
In addition, as the clinical usage of Ga is limited due to its
toxicity, we examined the toxicity profile of the combination of
Ga and liposomes only as gentamicin had no toxic effects on
lung cells.6,39 Interestingly, the Ga toxicity profile completely
changed when liposomes were involved and cell viability
increased compared with free Ga. A plausible explanation for
the latter could be the nature of the liposomes’ phospholipid
Free Ga
bilayers and their intimate communication with the lung cells,40
Lipo-Ga which might prevent the engulfed Ga within liposome bilayers

from direct contact with the lung cells.

60 *** In conclusion, we report a new strategy for liposomes as a
40
drug carrier by delivering two agents at the same time, which
optimized gentamicin and reduced the Ga toxicity in a success-
20 ful effort to prevent biofilm formation and bacterial resistance
that are associated with pulmonary chronic infection of CF
0 patients in vitro. However, an animal model is required to inves-
tigate this formulation capability in vivo.
l

15

30

60
ro

7.
nt
Co

Gallium (mM)
Figure 3. Effect of Lipo-Ga formulation on cell viability: the percentage
viability of A549 lung cells was measured after 24 h of incubation with free
Ga (white bars) and Lipo-Ga (black bars) at 7.5, 15, 30 and 60 mM Ga.
Untreated A549 cells acted as a positive control. Data represent the means +
SEM of three independent experiments. P values compare free Ga with
Lipo-Ga; **P  0.01 and ***P  0.001.
Acknowledgements
All clinical isolates were kindly provided by the Department of
Microbiology, Memorial Hospital, Sudbury, Ontario, Canada.
Also, we thank Dr Clay Fuqua from the Department of Biology
at Indiana University (Bloomington, IN, USA) for providing
A. tumefaciens strains.

1295
 Halwani et al.
Funding
This work was supported, in part, by a research grant from the
Ministry of Health of Saudi Arabia (M. H.) and the Laurentian
University Research Funds (A. O.).
Transparency declarations
None to declare.
References
1. Moskowitz SM, Gibson RL, Effmann EL. Cystic fibrosis lung
disease: genetic influences, microbial interactions, and radiological
assessment. Pediatr Radiol 2005; 35: 739– 57.
2. Zeitlin PL. Emerging drug treatments for cystic fibrosis. Expert
Opin Emerg Drugs 2007; 12: 329– 36.
3. Pukhalsky AL, Kapranov NI, Kalashnikova EA et al. Inflammatory
markers in cystic fibrosis patients with lung Pseudomonas aeruginosa
infection. Mediators Inflamm 1999; 8: 159–67.
4. Sermet-Gaudelus I, Renouil M, Fajac A et al. In vitro prediction
of stop-codon suppression by intravenous gentamicin in patients with
cystic fibrosis: a pilot study. BMC Med 2007; 5: 5.
5. Wilschanski M, Yahav Y, Yaacov Y et al. Gentamicin-induced
correction of CFTR function in patients with cystic fibrosis and CFTR
stop mutations. N Engl J Med 2003; 349: 1433– 41.
6. Scott CS, Retsch-Bogart GZ, Henry MM. Renal failure and
vestibular toxicity in an adolescent with cystic fibrosis receiving
gentamicin and standard-dose ibuprofen. Pediatr Pulmonol 2001; 31:
314–6.
7. Spencker FB, Staber L, Lietz T et al. Development of resistance
in Pseudomonas aeruginosa obtained from patients with cystic fibrosis
at different times. Clin Microbiol Infect 2003; 9: 370– 9.
8. Alkawash MA, Soothill JS, Schiller NL. Alginate lyase enhances
antibiotic killing of mucoid Pseudomonas aeruginosa in biofilms.
APMIS 2006; 114: 131– 8.
9. Cunha MV, Sousa SA, Leitao JH et al. Studies on the involve-
ment of the exopolysaccharide produced by cystic fibrosis-associated
isolates of the Burkholderia cepacia complex in biofilm formation and in
persistence of respiratory infections. J Clin Microbiol 2004; 42:
3052–8.
10. Waters CM, Lu W, Rabinowitz JD et al. Quorum sensing controls
biofilm formation in Vibrio cholerae through modulation of cyclic
di-GMP levels and repression of vpsT. J Bacteriol 2008; 190:
2527–36.
11. Schaber JA, Triffo WJ, Suh SJ et al. Pseudomonas aeruginosa
forms biofilms in acute infection independent of cell-to-cell signaling.
Infect Immun 2007; 75: 3715– 21.
12. Mugabe C, Halwani M, Azghani AO et al. Mechanism of
enhanced activity of liposome-entrapped aminoglycosides against
resistant strains of Pseudomonas aeruginosa. Antimicrob Agents
Chemother 2006; 50: 2016– 22.
13. Sachetelli S, Khalil H, Chen T et al. Demonstration of a fusion
mechanism between a fluid bactericidal liposomal formulation and
bacterial cells. Biochim Biophys Acta 2000; 1463: 254– 66.
14. Clarke TE, Tari LW, Vogel HJ. Structural biology of bacterial iron
uptake systems. Curr Top Med Chem 2001; 1: 7 –30.
15. Neilands JB. Siderophores: structure and function of microbial
iron transport compounds. J Biol Chem 1995; 270: 26723–6.
16. Kaneko Y, Thoendel M, Olakanmi O et al. The transition metal
gallium disrupts Pseudomonas aeruginosa iron metabolism and has
antimicrobial and antibiofilm activity. J Clin Invest 2007; 117: 877–88.
1296
17. Harrington JR, Martens RJ, Cohen ND et al. Antimicrobial
activity of gallium against virulent Rhodococcus equi in vitro and in
vivo. J Vet Pharmacol Ther 2006; 29: 121–7.
18. Halwani M, Mugabe C, Azghani AO et al. Bactericidal efficacy of
liposomal aminoglycosides against Burkholderia cenocepacia.
J Antimicrob Chemother 2007; 60: 760–9.
19. Alipour M, Omri A, Smith MG et al. Prophylactic effect of liposo-
mal N-acetylcysteine against LPS-induced liver injuries. J Endotoxin
Res 2007; 13: 297 –304.
20. Halwani M, Blomme S, Suntres ZE et al. Liposomal
bismuth-ethanedithiol formulation enhances antimicrobial activity of
tobramycin. Int J Pharm 2008; 358: 278–84.
21. Abdi-Ali A, Mohammadi-Mehr M, Agha Alaei Y. Bactericidal
Downloaded from https://academic.oup.com/jac/article-abstract/62/6/1291/773926 by guest on 28 November 2019
activity of various antibiotics against biofilm-producing Pseudomonas
aeruginosa. Int J Antimicrob Agents 2006; 27: 196–200.
22. Agarwal G, Kapil A, Kabra SK et al. In vitro efficacy of cipro-
floxacin and gentamicin against a biofilm of Pseudomonas aeruginosa
and its free-living forms. Natl Med J India 2005; 18: 184 –6.
23. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms:
a common cause of persistent infections. Science 1999; 284:
1318–22.
24. Drulis-Kawa Z, Gubernator J, Dorotkiewicz-Jach A et al. In vitro
antimicrobial activity of liposomal meropenem against Pseudomonas
aeruginosa strains. Int J Pharm 2006; 315: 59 –66.
25. Ryan MA, Akinbi HT, Serrano AG et al. Antimicrobial activity of
native and synthetic surfactant protein B peptides. J Immunol 2006;
176: 416–25.
26. Das AR, Dattagupta N, Sridhar CN et al. A novel thiocationic
liposomal formulation of antisense oligonucleotides with activity
against Mycobacterium tuberculosis. Scand J Infect Dis 2003; 35:
168–74.
27. Pinto-Alphandary H, Andremont A, Couvreur P. Targeted deli-
very of antibiotics using liposomes and nanoparticles: research and
applications. Int J Antimicrob Agents 2000; 13: 155–68.
28. Zaru M, Mourtas S, Klepetsanis P et al. Liposomes for drug
delivery to the lungs by nebulization. Eur J Pharm Biopharm 2007; 67:
655–66.
29. Patriquin GM, Banin E, Gilmour C et al. Influence of quorum
sensing and iron on twitching motility and biofilm formation in
Pseudomonas aeruginosa. J Bacteriol 2008; 190: 662–71.
30. Meers P, Neville M, Malinin V et al. Biofilm penetration, triggered
release and in vivo activity of inhaled liposomal amikacin in chronic
Pseudomonas aeruginosa lung infections. J Antimicrob Chemother
2008; 61: 859–68.
31. Beaulac C, Clement-Major S, Hawari J et al. Eradication of
mucoid Pseudomonas aeruginosa with fluid liposome-encapsulated
tobramycin in an animal model of chronic pulmonary infection.
Antimicrob Agents Chemother 1996; 40: 665–9.
32. Bonte F, Juliano RL. Interactions of liposomes with serum
proteins. Chem Phys Lipids 1986; 40: 359–72.
33. Simoes S, Moreira JN, Fonseca C et al. On the formulation of
pH-sensitive liposomes with long circulation times. Adv Drug Deliv Rev
2004; 56: 947–65.
34. Hernandez-Caselles T, Villalain J, Gomez-Fernandez JC.
Influence of liposome charge and composition on their interaction
with human blood serum proteins. Mol Cell Biochem 1993; 120:
119–26.
35. Sun WQ, Leopold AC, Crowe LM et al. Stability of dry liposomes
in sugar glasses. Biophys J 1996; 70: 1769–76.
36. Peeters E, Nelis HJ, Coenye T. Resistance of planktonic and
biofilm-grown Burkholderia cepacia complex isolates to the transition
metal gallium. J Antimicrob Chemother 2008; 61: 1062–5.
 Liposomal-gallium-gentamicin formulation
37. Aslangul E, Massias L, Meulemans A et al. Acquired gentamicin 39. Russell BH, Vasan R, Keene DR et al. Potential dissemination
resistance by permeability impairment in Enterococcus faecalis. of Bacillus anthracis utilizing human lung epithelial cells. Cell Microbiol
Antimicrob Agents Chemother 2006; 50: 3615– 21. 2008; 10: 945–57.
38. Singh PK, Schaefer AL, Parsek MR et al. Quorum-sensing 40. Desjardins A, Chen T, Khalil H et al. Differential behaviour of
signals indicate that cystic fibrosis lungs are infected with bacterial fluid liposomes toward mammalian epithelial cells and bacteria: restric-
biofilms. Nature 2000; 407: 762 –4. tion of fusion to bacteria. J Drug Target 2002; 10: 47–54.

Downloaded from https://academic.oup.com/jac/article-abstract/62/6/1291/773926 by guest on 28 November 2019

1297