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JAC Advance Access published online on October 18, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn422
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© 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

Original research

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

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

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


   Abstract
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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 gentamicin 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 gentamicin alone in eradicating antibiotic-resistant P. aeruginosa isolates growing in a planktonic or biofilm community.

Key Words: antibiotics , aminoglycosides , biofilms , quorum sensing , toxicity


   Introduction
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Cystic fibrosis (CF) is a chronic disease with no cure available at the present time. However, efforts in the development of gene therapy to restore the CF transmembrane conductance regulator (CFTR) function might improve outcomes.1 Equally important is the improvement of existing therapeutics to prevent emerging antibiotic-resistant bacteria common to CF.2 CFTR chloride channel deficiency or absence results in an abnormal epithelia lining fluid suitable for microbial colonization and growth.3 Aminoglycoside antibiotics such as gentamicin are not only highly effective against Pseudomonas aeruginosa but can also suppress the expression of some of the nonsense mutations of the CFTR, hence allowing it to function normally.4,5 However, gentamicin use is restricted due to the emerging resistant strains.6,7 Bacteria can become resistant by changing their outer membrane permeability, an effect attributed to the alterations in exopolysaccharide and alginate biofilm production,8,9 which is controlled by bacterial communication through quorum sensing (QS) signal molecules.10,11

Liposomes, as a drug carrier system, have the capacity to optimize antibiotic therapy by reducing antibiotic toxicity, improving drug uptake and enhancing bactericidal efficacy through fusion with the bacterial membrane.12,13 Recently, gallium has emerged as an effective inhibitory agent against P. aeruginosa growth and biofilm formation. Disruption of iron metabolism increases the vulnerability of most infecting bacteria because iron is essential for growth and the functioning of key enzymes, such as those involved in protein and DNA synthesis, electron transport and oxidative stress.14,15 It is recognized that Ga, due to its chemical similarities to Fe, can substitute for Fe in many biological systems and inhibits Fe-dependent processes.16,17

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 formulation, 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.


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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 isolated 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 37°C 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 –80°C, supplemented with 10% glycerol, and subcultured for 18 h in Mueller–Hinton broth prior to experiments. Agrobacterium tumefaciens strains A136 (Ti–) (pCF218) (pCF372) and KYC6 were used in the QS experiments and cultured in Luria–Bertani (LB) broth at 30°C, supplemented with spectinomycin (50 mg/L), tetracycline (4.5 mg/L) and 15% glycerol for storing at –80°C. Routine QS experiments were carried out by subculturing 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 multilamellar 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 µM 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 rehydrate, sterile water (10% of final volume before lyophilization) was added, and the mixtures were incubated at 50°C for 30 min. This step was repeated once more with PBS. Additional PBS was then 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 4°C 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 diameter 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 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 37°C and then inoculated into the holes of the agar plates containing S. aureus ATCC 29213 in duplicate, as well as 20 µM Ga as a control. Plates were incubated at 37°C for 24 h, and the inhibition zones were then measured. EE was calculated as follows:


Formula

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 transferred 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 25°C to release the liposomal content. Samples were then subjected to heated sand bath digestion at 135–140°C 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 analysed 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, 4°C) and gentamicin concentrations 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 gentamicin.20 Briefly, strains of S. aureus and P. aeruginosa were exposed to serial dilutions of free gentamicin or Lipo-Ga-GEN. The contribution of Ga to the MICs was assessed by exposing the same bacterial strains to different concentrations of Ga added in the forms of free Ga, Ga-GEN, Lipo-Ga and Lipo-Ga-GEN, with a starting concentration of 42 µM. 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 incubated for a further 24 h at 37°C to determine the MBCs.

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 37°C (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 37°C. Aliquots of 100 µL of each sample were then plated on Mueller–Hinton agar and incubated for 24 h at 37°C. 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 37°C. Samples of PBS-treated P. aeruginosa were used as positive controls. The samples were then centrifuged for 15 min (18 000 g) at 4°C; the pellets were discarded and the supernatants were assayed for AHL production by the following procedure: mixed sterilized LB agar with A. tumefaciens A136 (Ti–) (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 37°C, the edge of the holes was inspected for a blue pigmentation, an AHL indicator. Samples of A136 supernatant served as negative controls as the overexposure 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 37°C for 24 h was compared with that of the samples treated with the lowest concentration of Lipo-Ga-GEN that prevented AHL production.

Lipo-Ga toxicity study

A549 cells at a density of 5 x 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 µM 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 µM Lipo-Ga-GEN) and different concentrations of free gentamicin (10–1000 mg/L). All dishes were incubated for 24 h in 5% CO2 at 37°C. 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 4°C. 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 experiments. Comparisons between individual columns and groups were made by paired Student's t-test, and *P ≤ 0.05, **P ≤ 0.01 or ***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.


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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 incorporated into the Lipo-Ga-GEN formulation was 19 ± 0.15 µM, as determined by atomic absorption analysis. Lipo-Ga-GEN retained >92% of gentamicin in PBS at 4°C and in BAL at 37°C for 48 h. However, gentamicin was significantly released (43%) from the formulation in plasma for 48 h at 37°C (Figure 1).


Figure 1
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  		Figure 1. Lipo-Ga-GEN formulation stability in PBS at 4°C (open squares), in BAL at 37°C (filled circles) and in plasma at 37°C (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.

 
MIC and MBC determination

The MICs of Lipo-Ga-GEN for all P. aeruginosa strains examined 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 remarkable (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).


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  		Table 1. MIC comparison of the drug formulations

 


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  		Table 2. MBC comparison of the drug formulations

 
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 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.


Figure 2
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  		Figure 2. MBEC assay: antibacterial effect on PA-48912-1 (white bars) and PA-48912-2 (black bars). Lipo-Ga-GEN (0.6 µM 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 µM), Ga-GEN (0.6 µM and 8 mg/L), Lipo-Ga (0.6 µM) 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.

 
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 µM 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 µM 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).


Figure 3
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  		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 µM 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.

 

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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 ineffective in penetrating bacterial biofilm communities within the tissues or on medical devices.21,22 Bacterial biofilms are responsible 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 metabolism, important in the development of biofilm production.16,29 Recent studies have shown that liposomes can effectively penetrate 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 components such as lipoproteins, albumin, immunoglobulins and 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 liposomal structural integrity and permeability properties with leakage of the entrapped agents. It has been shown that liposomes containing only one class of negatively charged phospholipids 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 gentamicin, free Ga and the combination Ga-GEN were not as effective at reducing the rate of the P. aeruginosa antibiotic resistance development phenomenon. This finding is consistent with the results reported by Peeters et al.,36 who showed that Ga had a limited effect on Burkholderia cepacia strains in the presence or absence of iron; impermeability of those strains to gentamicin was due to changes in LPS structure.36,37 However, we observed a slight enhancement of gentamicin bacterial killing when incorporated within liposomes due to their fusible property with bacterial membranes.12 On the other hand, Lipo-Ga-GEN inhibited the growth of gentamicin-resistant strains of P. aeruginosa and also enhanced Ga uptake, which clearly supports our hypothesis of using two antibacterial agents.

Furthermore, in the in vitro model of P. aeruginosa biofilm, the biofilm bacteria were aggressively resistant to free gentamicin, Ga and the combination of Ga-GEN without any eradication 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 formation.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 bilayers and their intimate communication with the lung cells,40 which might prevent the engulfed Ga within liposome bilayers from direct contact with the lung cells.

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


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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.).


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None to declare.


   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.


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