(M1430-01-03) Fusogenic Liposomes as an Approach to Enhance the Efficacy of Antimicrobials

Purpose: The global mortality due to antimicrobial resistance (AMR) was estimated as 1.27 million deaths in 2019 and is expected to reach 10 million by 2050 (1, 2). The pace of new antimicrobials development does not cope with the alarmingly rapid emergence of resistant microorganisms. However, novel formulation approaches can contribute to tackling the threat and improving the efficacy of conventional antimicrobial agents. Among the reported hindrances for antimicrobial activity, is the ability to penetrate the microbial cell envelope, which includes cell membrane, cell wall and outer membrane in the case of gram-negative bacteria (3, 4). For instance, vancomycin is only effective against gram-positive bacteria as it is unable to penetrate the outer membrane of gram-negative bacteria. Thus, using fusogenic liposomes can help by broadening the antibiotics spectrum of activity (5, 6). In this work, positively charged fusogenic liposomes was developed as a drug delivery system to facilitate antimicrobials penetration through microbial cell envelope. The prepared liposomes were tested against microorganisms representing the different structural cell envelopes of fungi, gram-positive and gram-negative bacteria.

Methods: Positively charged fusogenic liposomes were prepared using film hydration/extrusion method with DPPC:DOPE:DOTAP in 5:4:1 molar ratio. The physical stability of the liposomes was monitored for one year by measuring the size, polydispersity index (PDI) and zeta potential at different time points. Escherichia coli, Staphylococcus aureus and Candida albicans were selected to study liposomal penetration. Cellular uptake of liposomes was measured with two different methods, lipid mixing assay and flow cytometer assay. In the former method, liposomes were labelled with two fluorescent probes (lissamine rhodamine and NBD) and the measurements were based on fluorescence resonance energy transfer (FRET) phenomenon using a fluorescence microplate reader. In the flow cytometer assay, liposomes were labelled with PKH67 fluorescent prob and after incubation with cells, samples were measured using a flow cytometer and number of fluorescent cells was counted. Additionally, the biocompatibility of the prepared liposomes was determined using a haemolysis assay. Different concentrations of liposomes were incubated with red blood cells in phosphate buffered saline. Samples were centrifuged at 4000 rpm for 10 minutes, then supernatants collected, and haemoglobin spectrophotometrically determined at 405 nm.

Results: We were able to prepare nanosized fusogenic liposomes with a size of 135.3 ± 1.8 nm and a PDI of 0.067 ± 0.013. The liposomes were stable for one year at 4°C despite the devoid of the composition from cholesterol. The measured zeta potential was 25.7 ± 1.01 mV, which may explain the significant one-year stability of the liposomes. The extent of liposomal fusion with microorganisms measured with lipid mixing assay was 15.06%, 13.05% and 9.83% with E. coli, S. aureus and C. albicans, respectively. Fusion measured using flow cytometer assay was 7.7% and 2.1% with E. coli and S. aureus, respectively. Meanwhile, no uptake was detected with C. albicans using this method, which could be justified by the lower sensitivity of the method. Higher degree of fusion with E. coli can be due to the structural resemblance of liposomes and the outer membrane of gram-negative bacteria, which is mainly composed of phospholipids. Finally, the haemolysis assay result demonstrates that the prepared fusogenic liposomes are biocompatible in concentrations up to 10 mM.

Conclusion: The prepared fusogenic liposomes showed acceptable physical properties and long-term stability. The liposomes demonstrated the ability to fuse with microorganisms and were biocompatible with red blood cells. Consequently, the developed liposomal formulation could be a promising drug delivery system to improve the efficacy of antimicrobial agents. Which needs to be confirmed in future work by drug-loading and observing the effect on microbiological activity.

References: 1. Murugaiyan J, Kumar PA, Rao GS, Iskandar K, Hawser S, Hays JP, et al. Progress in Alternative Strategies to Combat Antimicrobial Resistance: Focus on Antibiotics. Antibiotics. 2022;11(2):200.
2. Mestrovic T, Robles Aguilar G, Swetschinski LR, Ikuta KS, Gray AP, Davis Weaver N, et al. The burden of bacterial antimicrobial resistance in the WHO European region in 2019: a cross-country systematic analysis. The Lancet Public Health. 2022;7(11):e897-e913.
3. Lambert P. Cellular impermeability and uptake of biocides and antibiotics in Gram‐positive bacteria and mycobacteria. Journal of applied microbiology. 2002;92(s1):46S-54S.
4. Santos RS, Figueiredo C, Azevedo NF, Braeckmans K, De Smedt SC. Nanomaterials and molecular transporters to overcome the bacterial envelope barrier: Towards advanced delivery of antibiotics. Advanced Drug Delivery Reviews. 2018;136-137:28-48.
5. Nicolosi D, Scalia M, Nicolosi VM, Pignatello R. Encapsulation in fusogenic liposomes broadens the spectrum of action of vancomycin against Gram-negative bacteria. International journal of antimicrobial agents. 2010;35(6):553-8.
6. Pignatello R, Nicolosi D, Nicolosi VM. Fusogenic liposomes as new carriers to enlarge the spectrum of action of antibiotic drugs against Gram-negative bacteria. Science Against Microbial Pathogens: Communicating Current Research and Technological Advances Badajoz, Spain: Formatex Research Center. 2011:52-60.

Acknowledgements: No conflict of interest

Size and polydispersity index (PDI) measurements of the prepared liposomal formulation presented as mean ± SD (n=3) at different time points during one year of monitoring.