Cholesterol-Rich Antibiotic-Loaded Liposomes as Efficient Antimicrobial Therapeutics

Evangelos Natsaridis,1,2,* Foteini Gkartziou,2,* Panagiota Mouzoura,1 Spyridon Mourtas,3 Konstantina Papadia,1 Fevronia Kolonitsiou,4 Pavlos Klepetsanis1,2 ,† Catherine R Dermon,5 Iris Spiliopoulou,4,6 Sophia G Antimisiaris1,2 1Laboratory of Pharmaceutical Technology, Department of Pharmacy, School of Health Sciences, University of Patras, Rion, Patras, 26504, Greece; 2Institute of Chemical Engineering Sciences, FORTH/ICE-HT, Platani, Patras, 26504, Greece; 3Department of Chemistry, University of Patras, Rion, Patras, 26510, Greece; 4Department of Microbiology, School of Medicine, University of Patras, Rion, Patras, 26504, Greece; 5Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, Rion, Patras, 26504, Greece; 6National Reference Centre for Staphylococci, School of Medicine, University of Patras, Rion, Patras, 26504, Greece†Pavlos Klepetsanis passed away on October 23, 2024*These authors contributed equally to this workCorrespondence: Sophia G Antimisiaris, Laboratory Pharm Technology, University of Patras, Pharmacy Building, Rio Campus, Rio Patras, 26504, Greece, Tel +30-2610-962332, Email [email protected]: Liposomal antibiotics have demonstrated higher bacteriostatic and bactericidal activities than free drugs. In this study, we investigated the effects of cholesterol (Chol) content of liposomes, liposome concentration, and surface coating with polyethylene glycol (PEG) on the antimicrobial activity of moxifloxacin (MOX) liposomes against Staphylococcus epidermidis (ATCC 35984) (S.e).Methods: MOX-liposome compositions with increasing Chol content were evaluated for their susceptibility to planktonic S.e (growth inhibition, killing, and live-dead staining), as well as against pre-formed biofilms (crystal violet, MTT assay, and confocal microscopy). The MOX-liposomes prepared by active loading were characterized in terms of loading, size distribution, and zeta potential.Results-Discussion: All liposomes had nano-dimensions ranging in diameter from 92nm to 114nm, with zeta-potential values from − 2.30mV to − 4.50mV. Planktonic bacteria and established biofilms are significantly more susceptible to MOX-liposomes with higher Chol-content than other liposome-types, and the same MOX dose encapsulated in 10 times higher lipids demonstrated higher antimicrobial activity. Coating the MOX liposomes with PEG did not affect their activity. Flow cytometry showed higher binding of Chol-rich liposomes to bacteria, explaining the higher antimicrobial activity. Interestingly, the integrity of calcein-loaded Chol-rich liposomes was much lower than that of liposomes with low or no Chol during incubation with various strains of S. epidermidis. In vivo results in a zebrafish infection model (bacteremia) confirmed the superior activity of Chol-rich MOX-liposomes compared to the free drug.Conclusion: The current in vitro and in vivo findings demonstrated the potential of PEGylated and Chol-rich liposomal antibiotics as highly efficient therapeutics for the treatment of S. epidermidis infections. Keywords: liposomes, cholesterol, antimicrobial, in vivo, antibiotic, moxifloxacin, Staphylococcus epidermidis