(L-463) Polymyxin B-modified Liposomal Ciprofloxacin for Active Targeting and Treatment of Pseudomonas aeruginosa Infections

Antimicrobial resistance is a severe threat to human health. According to the Centers of Disease Control and Prevention, 2.8 million infections are caused by resistant microbes, leading to 56,000 deaths annually in the U.S. alone. Gram-negative bacteria are increasingly resistant to multiple antibiotics, with Pseudomonas aeruginosa being one of three “highest priority” pathogens.1 There is a lack of FDA-approved drug delivery platforms that target Gram-negative bacteria, including P. aeruginosa. Novel drug delivery systems must be developed to directly target these infections and enhance the treatment efficacy of existing antibiotics. Liposomes are lipid-based nanoparticles that can encapsulate both hydrophobic and hydrophilic drugs and passively accumulate at the infection site due to the enhanced permeability and retention (EPR) effect.2 Liposomes can also be functionalized with different surface ligands for enhanced drug delivery via active targeting of bacteria.3 Polymyxin B (PMB) is a polypeptide antibiotic that has a high affinity for lipid A of lipopolysaccharide, which is found in the outer membrane of Gram-negative bacteria, including P. aeruginosa. The aim of this study is to take advantage of the targeting capability of polymyxin B for the bacterial membrane to develop polymyxin B-modified liposomes (PMB-Lipo) to target P. aeruginosa infections and increase antibiotic delivery to the infection site.

To create the targeting ligand (PMB conjugate), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG2000-COOH) was covalently conjugated to PMB via amide formation using the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysulfosuccinimide (sulfo-NHS) coupling method. Unconjugated PMB was removed using a dialysis cassette (3.5 kDa MWCO). Successful conjugation was confirmed via proton nuclear magnetic resonance (1H-NMR) spectroscopy. The PMB conjugate was then incorporated into hydrogenated soy phosphatidylcholine (HSPC) liposomes (PMB-Lipo) via the lipid film hydration method with a molar ratio of 58:1:1:40 (HSPC:PEG2000-PE:DSPE-PEG2000-PMB:cholesterol). Unmodified HSPC liposomes (Lipo) were prepared as a control group. Both Lipo and PMB-Lipo were tagged with 0.1% (w/w) rhodamine B, a fluorescent molecule. The hydrodynamic diameter, PDI, and zeta potential of both liposomes were measured by dynamic light scattering (DLS). The targeting and antibacterial capabilities of PMB-Lipo were investigated using P. aeruginosa PA01. For the targeting assay, PMB-Lipo and Lipo were mixed with PA01 in saline and left shaking for 30 minutes at 37 ℃. After incubation, the mixture was centrifuged and the pellet was resuspended in phosphate buffered saline (PBS 1 ✕, pH 6.8). The fluorescence intensity of rhodamine B (excitation/emission: 516 nm/580 nm) was measured for both the resuspended pellet and supernatant as an indicator of liposomes attached to the cells and free liposomes, respectively. The bacteria-liposome interactions were also visualized using confocal microscopy. The minimum inhibitory concentration (MIC) of PMB-Lipo was also investigated using the microdilution assay method (MDA). All MIC values are reported with respect to the equivalent PMB concentration.

PMB was covalently conjugated to the lipid DSPE-PEG2000-COOH via EDC-mediated amide formation. 1H-NMR spectra of DSPE-PEG2000-COOH, PMB, and DSPE-PEG2000-PMB confirmed successful conjugation (Figure 1). PMB-Lipo and Lipo were fabricated with and without the DSPE-PEG2000-PMB conjugate, respectively (Figure 2). Lipo and PMB-Lipo exhibited similar hydrodynamic diameters of ~116 and ~104 nm, respectively (Figure 3a). Additionally, Lipo and PMB-Lipo exhibited zeta-potentials of −0.4 mV and 2.1 mV, respectively (Figure 3b). The slight increase in charge of PMB-Lipo was expected due to the positive charges of PMB. To investigate the interaction of the rhodamine B-tagged liposomes with PA01, we examined the fluorescence intensity of the liposomes upon incubation with PA01. We found that the resuspended pellet of PA01 that had been incubated with PMB-Lipo exhibited 32% greater fluorescence intensity than those incubated with Lipo (Figure 4a), suggesting greater interaction of PMB-Lipo with PA01. The confocal microscopy (Figure 4b,c), also showed greater fluorescence in the PMB-Lipo incubated cells, again confirming greater interaction of these liposomes with the bacteria than control Lipo formulations. The antibacterial activity of PMB-Lipo was assessed and compared to that of free PMB, the PMB conjugate, and Lipo. Free PMB exhibited a MIC of 2.5 µg/mL, while the PMB conjugate demonstrated a MIC of 5 µg/mL. Additionally, PMB-Lipo exhibited a MIC of 10 µg/mL (Figure 5). Lipo did not exhibit any antibacterial activity (Data not shown). With the promising interaction of PMB-Lipo observed here, we will load the PBM-Lipo with ciprofloxacin, a broad spectrum fluoroquinolone antibiotic effective against P. aeruginosa, and investigate their antibacterial efficacy. We hypothesize that the PMB-Lipo will enhance the activity of the loaded ciprofloxacin both due to increasing interaction with bacterial cells and also the inherent antibacterial activity of these liposomes. We will examine the impact of antibacterial resistance development upon exposure to these liposome formulations.

1Hart, R. J., & Morici, L. A., 2022, Front. Microbiol., 13, 870104.

2Sercombe, L., et. al., 2015, Front. Pharmacol., 6, 286.