The rise of antibiotic resistance threatens our ability to treat even minor bacterial infections. With few new drugs in development and rates of resistance increasing, we urgently need new approaches to fight bacterial infections. The goal of this project is to develop a biologically inspired drug delivery vehicle to enable the controlled release of antibiotics. Such a system will permit the use of currently available antibiotics in a more sustainable manner and will overcome certain mechanisms of resistance. Gram-negative bacteria are particularly difficult to treat with antibiotics due to their dual-membrane cell wall, as well as other resistance mechanisms including overexpression of efflux pumps and enzymatic degradation of the drug. In this work, we propose to take advantage of a natural system used by bacteria to deliver biomolecules to other bacterial cells called outer membrane vesicles (OMVs). These OMVs have been demonstrated to deliver functional cargo to the cytosol of Gram-negative bacteria. While this mechanism has not yet been fully uncovered, it suggests the possibility of using OMVs for antibiotic delivery, particularly to Gram-negative bacteria. OMVs provide a number of advantages as a drug delivery system, including stability, long-term in vivo delivery, and the ability to deliver molecules to bacterial cells, including Gram-negative cells. However, they suffer from certain limitations, including loading efficiency and the ability to readily alter membrane properties to improve function (“tunabilityâ€). In contrast, liposomes are readily loaded with cargo and their physical properties can be easily tuned with simple adjustments in lipid composition. We therefore hypothesized that a vehicle created from both liposomes and OMVs might facilitate antibiotic delivery and enhance activity in antibiotic-resistant strains of bacteria.
Liposomes of varying lipid composition were mixed with Escherichia coli OMVs and extruded to create semi-synthetic OMVs (SS-OMVs). We have demonstrated, using a dye dilution assay, that upon extrusion, the liposomes and OMVs mix thoroughly to create a hybrid vehicle. We verified using dynamic light scattering that the SS-OMVs are stable for at least seven days under a range of storage conditions. In addition, we showed that the SS-OMVs retain their luminal content over this time frame. Using a combination of membrane- and content-mixing assays, we have demonstrated that the SS-OMVs are able to fuse with Pseudomonas aeruginosa (PAO1) cells, and current work is focused on optimizing this delivery process. Together, these results demonstrate the potential of a new hybrid antibiotic delivery vehicle for the controlled delivery of antibiotics.
