Saleem, Naveed; (2024) Liposomal antibiotics enhance bacterial killing and ameliorate systemic inflammation. Masters thesis (M.Phil), UCL (University College London).

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Abstract

INTRODUCTION The rising incidence of resistant organisms, particularly Gram-negative, is a global threat. The development of novel antibiotics is limited; hence modification of existing antibiotics is an attractive option. Positively charged liposomes are preferentially attracted to negatively-charged Gram-negative bacterial membranes via ionic interactions and could be employed to deliver existing antibiotics. This may help to overcome multidrug-resistant bacterial infections by achieving higher target concentrations, as well as facilitating reductions in antibiotic dosing thereby limiting drug toxicity. METHODS Meropenem was encapsulated in cationic liposomes by combining phosphatidylcholine (PC), 2,3–di-methyl dioctadecyl ammonium bromide (DODAB) and cholesterol (Chol). Liposome characterization was quantified using Dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC). Encapsulation efficacy and total drug concentrations were calculated for different non-PEGylated and PEGylated meropenem-encapsulated cationic liposomal preparations. The in-vitro antimicrobial activity of free and meropenem-encapsulated cationic liposomes with or without PEG lipids was assessed using 24-hour broth microdilution, through determination of minimum inhibitory concentration (MIC) and plotting growth curves and inhibition kinetics. To assess liposomal safety, whole blood drawn from healthy volunteers was incubated with standard meropenem and non-PEGylated and PEGylated meropenem-encapsulated cationic liposomes at different therapeutic doses for 6 hours. Leucocyte toxicity was assessed using fluorescence-activated cell sorting (FACS) with staining for reactive oxygen species production and cell viability. RESULTS In-vitro drug susceptibility findings Non-PEGylated and PEGylated meropenem-encapsulated cationic liposomal formulations were associated with enhanced in-vitro bactericidal activity. In an in-vitro model, up to 30-fold reduction in MIC (minimum inhibitory concentration) was achieved against multiple laboratory and clinical strains of Gram-negative bacteria meropenem-encapsulated cationic liposomes compared to native meropenem. In-vitro biocompatibility findings 19 Meropenem-encapsulated liposomes with PEGylation prevented immune cell activation i.e., leukocyte internalization, ROS production, and death cell, pro-inflammatory cytokines in comparison to free standard meropenem irrespective of drug concentrations. CONCLUSION I have developed unique formulations of meropenem-encapsulated cationic liposomes with enhanced antimicrobial activity against different laboratory and clinicalstrains of Gram-negative bacteria, compared to non-liposomal meropenem, in-vitro. Additionally, I have found unique formulations that demonstrated safety against human erythrocytes and immune cells ex-vivo. Future work should refine the optimization of structurally modified liposomal formulations encapsulating different antibiotics to improve the efficacy and safety with minimal drug-related adverse events, to develop a therapeutic intervention for AMR.

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