Delivery of Liposomal Contents to Outer Membrane Vesicles from Gram Negative Bacteria

Abstract

Gram negative bacteria produce small ∼50-200 nm vesicles from their outer membranes. These outer membrane vesicles (OMV) have been implicated in activities such as transmission of virulence factors, horizontal gene transfer and development of biofilms. In this investigation, we continue our studies on the association and/or fusion of various liposomes with OMV. The delivery of large encapsulated molecules into OMV from L. enzymogenes C3 was investigated using liposomes with lipid compositions previously observed to be apparently fusogenic (Bartos et al., Biophys. J. 104(2) suppl1, 90a). Liposomes (100 nm) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) in a 1:3 ratio or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were used to encapsulate dextran conjugates of Texas red averaging 40 kDa. They were incubated with Lysobacter OMV (30°C, 1 hr.), then sedimented through 15% iodixanol, and the fluorescence monitored as indicatiive of transfer of liposomal contents to fused products. Both liposomal compositions showed significant evidence of dextran transfer. Because biofilms also contain OMV, the interaction of these liposomes with E. coli (DH10B) biofilms was also investigated via fluorescence microscopy. Significant penetration and binding within the biofilm mass was observed, as well as possible fusion with OMV, and rarely, evidence of transfer of dextran into whole bacterial cells. Fluorescence resonance energy transfer (FRET)-based assays also demonstrated that liposomes as small as 30 nm could rapidly fuse with Lysobacter OMV, suggesting possible delivery to OMV with smaller perturbation and better biofilm penetration.