Liposomes as delivery vectors for nucleic acid mimics into Escherichia coli: effect of composition and...

The growing threat of antimicrobial resistance (AMR) requires the development of novel therapeutic solutions and Nucleic Acid Mimics (NAMs) arepresent a promising alternative. NAMs can hybridize with intracellular bacterial mRNA, working as antisense oligonucleotides (ASOs), being therefore potentially applied as targeted therapeutics (ASObiotics). Because NAMs do not naturally penetrate the bacterial envelope, effective delivery vectors are required. Liposomes represent a promising strategy to overcome this barrier, but their interaction with bacterial envelopes remains poorly understood. Here, we evaluated multiple liposomal formulations as delivery vectors for Cy3-labelled LNA/2'-O-methyl NAMs, targeting the essential acpP bacterial gene. Liposomes were engineered using different pairings of cationic/ionizable and helper lipids, a PEG-conjugated stabilizing lipid, and anti-Escherichia coli (E. coli) polyclonal antibodies. A DOTAP/DOPE formulation at 75:25 M ratio achieved over 80% delivery efficiency in E. coli. However, the inclusion of DSPE-methoxy-PEG resulted in reduced efficiency. The conjugation of anti-E. coli polyclonal antibodies significantly enhanced species-specific delivery, reducing labeling efficiency in non-target bacteria by approximately 20%. Higher antibody amounts improved delivery efficiency and specificity. The formulations showed no detectable cytotoxicity in vivo using the Galleria mellonella infection model. These findings demonstrate that antibody-functionalized liposomes provide an effective and selective platform for NAM delivery to bacterial cells.