Physicochemical characterization of liposomal aminoglycosides and the evaluation of their interactions with Pseudomonas aeruginosa isolated from a cystic fibrosis patient

The antimicrobial efficacy of liposomal drug delivery systems is more pronounced than that of free drugs as indicated by previous studies carried out in our laboratory. However, the relationship between the properties of liposome formulations and the delivery efficiency of tiposomal vesicles into Pseudomonas aeruginosa cells remains unclear. Therefore, we proposed that different liposomal compositions directly affect the phase properties of liposomal vesicles. The resulting phase characteristics of liposomal vesicles play a key role in determining the stability of aminoglycoside-containing liposomes, the extent of the interactions of liposomal vesicles with cells. and the rate and amount of the liposomal content translocated across the inner membrane of the bacterial cells.;In the second part of the study, cholesterol was incorporated into the DPPC vesicles. The physicochemical stability of DPPC/Chol liposomes containing gentamicin was investigated focusing on vesicle aggregation, domain separation, and gentamicin release from the vesicles. Gentamicin leaked out of the liposomes to a greater degree at 37°C than at 4°C. A large size distribution was generally observed over a 48-h time period. There was no significant size difference between the empty vesicles and the liposomal vesicles containing gentamicin. The fluidity, order parameter, and phase behavior of the phospholipid bilayers in response to the composition, temperature, and time were monitored over 48 h. While, our results revealed that gentamicin disturbs the packing and fluidizes the phospholipid chains, it did not seem to alter the nature of the microdomains at the polar interface of the bilayers.;Finally, three liposome formulations with different fluidity were prepared to study their interactions with a P. aeruginosa strain isolated from a cystic fibrosis patient. These formulations were composed of either DPPC alone, DPPC/Chol or DPPC/DMPG. It was found that the phase characteristics of liposomal vesicles governed the fusion between the vesicles and the cells, the translocation efficiency of the liposomal content across the inner membrane of P. aeruginosa, and the premature release of liposomal content outside the cells. The results showed that the DPPC vesicle fused with the bacterial cell to a greater extent, but DPPC/DMPG vesicles were more efficient in delivering their contents to the inner membrane of the bacteria. In conclusion, the interaction between the liposomal drugs with bacterial cells results in vesicle fusion and release of the liposomal content inside the cells, disruption of the bacterial membrane and subsequent uptake of the free drugs in close vicinity of the bacterial cells.;In the first part of the study, the impact of gentamicin (GN), tobramycin (TOB) and amikacin (AMI) on the thermodynamic properties of membrane bilayers composed of 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) was studied using a combination of differential scanning calorimetry (DSC), electron paramagnetic resonance (EPR) and 31p nuclear magnetic resonance (NMR) spectroscopy. The mole ratio of drug molecules residing inside to that outside of the vesicles was fixed at 1:0, 1:0.2, 1:1 and 1:2.5, respectively. Thermodynamic parameters, i.e., the main transition (Tm) temperature, transition enthalpy (DeltaHcal) and cooperatively (DeltaT 1/2), were extracted from the thermograms to characterize the effect of different drug concentration on the DPPC liposomes. The presence of the three drug molecules caused the Tm to increase and the DeltaH cal to decrease as the concentration of the antibiotics outside the vesicles increased. The drug molecules were believed to associate with the phospholipids via hydrogen bonding. It was found that the presence of GN, TOB and AMI antibiotics, at mole ratios of drug concentration inside the vesicles to that of outside ranging from 1:0 to 1:1, resulted in an increased fluidity (2Amax) and reduced order parameter (S) of the drug-containing liposomes. However, liposomal vesicles appeared to be more rigid and ordered as the drug concentration outside the vesicles were increased.