Targeted Disruption of the Outer Membrane of Gram-Negative Bacteria with Enediyne-Labeled Light-Responsive Au Nanorods

Today a significant number of Gram-negative bacteria have evolved to inactivate traditional antibiotics like penicillin and continue to develop resistance against next-generation antimicrobial drugs at a shocking rate. Taking inspiration from actinobacteria (order Actinomycetales) that make reactive enediynes with a (Z)-1,5-diyn-3-ene core to arm themselves, we report the photoactivation of the enediyne precursor octa-4-en-2,6-diyne-1,8-diamine (EDDA) to kill DH5α cells. To trigger Bergman cyclization of EDDA to make diradicals, we used gold nanorods (AuNRs) of chosen dimensions to act as a light-responsive, heat-generating platform. We put EDDA on AuNRs but also left room for polymyxin B nonapeptide (PMBN) to direct the PMBN/EDDA-loaded AuNRs to the surface of DH5α. PMBN interacts specifically with the lipopolysaccharide (LPS) component of the bacterial outer membrane. Importantly, LPS is highly conserved in the family of Gram-negative bacteria. After binding, we use low-power laser light (λ = 785 nm, 69 mW) to activate heat production at the gold metal surface. This heat triggers formation of a reactive 1,4-didehydrobenzene diradical intermediate that can abstract hydrogen atoms from proteins, lipids, and DNA to destroy their function. Transmission electron microscopy (TEM) shows that bifunctional PMBN/EDDA AuNRs are bound to the bacteria and that this interaction is dependent on the rod-to-cell ratio. Upon exposure to light, >85% of DH5α cells are killed according to LIVE/DEAD cell viability assays. The extent of cell death depends on the cell coverage density of bifunctional rods and the duration of exposure to light. Cell viability experiments were also performed in HEPES buffer. HEPES quenched diradical activity, resulting in unharmed cells. Together, our combined approach of photoactivation and EDDA-mediated radical formation demonstrates the potential of using activable enediynes to eliminate Gram-negative bacteria. In future work, it will be important to assess the effectiveness of this approach against other pathogenic organisms.