Activity and Interactions of Liposomal Antibiotics in Presence of Polyanions and Sputum of Patients with Cystic Fibrosis

BACKGROUND:To compare the effectiveness of liposomal tobramycin or polymyxin B against Pseudomonas aeruginosa in the Cystic Fibrosis (CF) sputum and its inhibition by common polyanionic components such as DNA, F-actin, lipopolysaccharides (LPS), and lipoteichoic acid (LTA). METHODOLOGY:Liposomal formulations were prepared from a mixture of 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) or 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) and Cholesterol (Chol), respectively. Stability of the formulations in different biological milieus and antibacterial activities compared to conventional forms in the presence of the aforementioned inhibitory factors or CF sputum were evaluated. RESULTS:The formulations were stable in all conditions tested with no significant differences compared to the controls. Inhibition of antibiotic formulations by DNA/F-actin and LPS/LTA was concentration dependent. DNA/F-actin (125 to 1000 mg/L) and LPS/LTA (1 to 1000 mg/L) inhibited conventional tobramycin bioactivity, whereas, liposome-entrapped tobramycin was inhibited at higher concentrations--DNA/F-actin (500 to 1000 mg/L) and LPS/LTA (100 to 1000 mg/L). Neither polymyxin B formulation was inactivated by DNA/F-actin, but LPS/LTA (1 to 1000 mg/L) inhibited the drug in conventional form completely and higher concentrations of the inhibitors (100 to 1000 mg/L) was required to inhibit the liposome-entrapped polymyxin B. Co-incubation with inhibitory factors (1000 mg/L) increased conventional (16-fold) and liposomal (4-fold) tobramycin minimum bactericidal concentrations (MBCs), while both polymyxin B formulations were inhibited 64-fold. CONCLUSIONS:Liposome-entrapment reduced antibiotic inhibition up to 100-fold and the CFU of endogenous P. aeruginosa in sputum by 4-fold compared to the conventional antibiotic, suggesting their potential applications in CF lung infections

Mechanism of Enhanced Activity of Liposome-Entrapped Aminoglycosides against Resistant Strains of Pseudomonas aeruginosa

Pseudomonas aeruginosa is inherently resistant to most conventional antibiotics. The mechanism of resistance of this bacterium is mainly associated with the low permeability of its outer membrane to these agents. We sought to assess the bactericidal efficacy of liposome-entrapped aminoglycosides against resistant clinical strains of P. aeruginosa and to define the mechanism of liposome-bacterium interactions. Aminoglycosides were incorporated into liposomes, and the bactericidal efficacies of both free and liposomal drugs were evaluated. To define the mechanism of liposome-bacterium interactions, transmission electron microscopy (TEM), flow cytometry, lipid mixing assay, and immunocytochemistry were employed. Encapsulation of aminoglycosides into liposomes significantly increased their antibacterial activity against the resistant strains used in this study (MICs of ≥32 versus ≤8 μg/ml). TEM observations showed that liposomes interact intimately with the outer membrane of P. aeruginosa, leading to the membrane deformation. The flow cytometry and lipid mixing assays confirmed liposome-bacterial membrane fusion, which increased as a function of incubation time. The maximum fusion rate was 54.3% ± 1.5% for an antibiotic-sensitive strain of P. aeruginosa and 57.8% ± 1.9% for a drug-resistant strain. The fusion between liposomes and P. aeruginosa significantly enhanced the antibiotics' penetration into the bacterial cells (3.2 ± 2.3 versus 24.2 ± 6.2 gold particles/bacterium, P ≤ 0.001). Our data suggest that liposome-entrapped antibiotics could successfully resolve infections caused by antibiotic-resistant P. aeruginosa through an enhanced mechanism of drug entry into the bacterial cells

Bactericidal activity and inhibition of tobramycin by DNA and F-actin.

<p>Bactericidal concentrations of free tobramycin (F-TOB), and liposomal tobramycin (L-TOB) at 2 mg/L were incubated with <i>P. aeruginosa</i> (ATCC 27853), or in presence of DNA/F-actin (125 to 1000 mg/L). Growth controls are represented at 0 h (empty bar), and 3 h (dark bar). Comparisons between free and liposomal tobramycin was made by ANOVA one-way post <i>t</i>-test, and <i>P</i>-values were considered significant when (***) <i>p</i><0.001.</p

Bactericidal activity and inhibition of antibiotics by DNA, F-actin, LPS and LTA.

<p>A) Bactericidal concentrations of free tobramycin (F-TOB) and liposomal tobramycin (L-TOB) were incubated in presence of LPS/LTA (1 to 1000 mg/L). B) Bactericidal concentrations of free polymyxin B (F-PMB) and liposomal polymyxin B (L-PMB) were incubated in presence of DNA/F-actin/LPS/LTA (125 to 1000 mg/L). Growth controls are represented at 0 h (empty bar), and 3 h (dark bar). Comparisons between free and liposomal formulations were made by ANOVA one-way post <i>t</i>-test, and <i>P</i>-values were considered significant when (**) <i>p</i><0.01, (***) <i>p</i><0.001.</p

Liposome-entrapped antibiotic stability assayed by microbiological assay.

<p>The stability of the liposomal formulations were examined at 37°C in an 18 h period in the presence of PBS, CAMH broth, supernatant of biofilm forming <i>P. aeruginosa</i>, a combination of DNA, F-actin, LPS, and LTA, and diluted intact or autoclaved sputum.</p

Bactericidal activity and inhibition of antibiotics by LPS and LTA.

<p>A) Bactericidal concentrations of free tobramycin (F-TOB) and liposomal tobramycin (L-TOB) were incubated in presence of LPS/LTA (1 to 1000 mg/L). B) Bactericidal concentrations of free polymyxin B (F-PMB) and liposomal polymyxin B (L-PMB) were incubated in presence of LPS/LTA (1 to 1000 mg/L). Growth controls are represented at 0 h (empty bar), and 3 h (dark bar). Comparisons between free and liposomal formulations were made by ANOVA one-way post <i>t</i>-test, and <i>P</i>-values were considered significant when (***) <i>p</i><0.001.</p

CF Sputum treatment with various antibiotic formulations.

<p>CFU counts were made after incubation of diluted CF sputum (1∶10 w/v) in PBS with two-fold dilutions of free tobramycin at 512 mg/L (F-TOB), liposomal tobramycin at 128 mg/L (L-TOB), free polymyxin B at 32 mg/L (F-PMB), and liposomal polymyxin B at 8 mg/L (L-PMB). Growth controls are represented at 0 h (empty bar), and 18 h (dark bar).</p

Minimum Bactericidal Concentrations.

<p>Bactericidal activity of free and liposomal formulations against susceptible <i>P. aeruginosa</i> ATCC 27853 strain was carried out in broth alone or in presence of DNA/F-actin/LPS/LTA at a final concentration of 1000 mg/L.</p