Abstract
Bacteriophages are promising alternative antimicrobial agents due to their high specificity for host bacteria and minimal immunogenicity in humans. However, their therapeutic application is limited by their nature as biological entities, with potential evolutionary consequences. In this study, we address these challenges by repurposing only the structural components of bacteriophages as vesicles to deliver antibiotics directly into the cytoplasm of bacterial hosts. This approach is based on two key hypotheses: first, antibiotics such as β-lactams remain effective against resistant bacteria if injected directly into the cytoplasm, bypassing resistance mechanisms; second, phage structures can be synthesized and self-assembled in vitro using modular genomes and cell-free protein expression to carry small molecules as cargo. To test these hypotheses, we utilized T7 phages and penicillin-resistant Escherichia coli as a model system. First, we designed the T7 phage genome into a modular format containing only the genes encoding structural components and synthesized the gene fragments via de novo gene synthesis. These phage structures were then rebooted in vitro using cell-free protein expression in the presence of penicillin G, allowing the antibiotics to be incorporated as cargo during the self-assembly process. Finally, we tested the antimicrobial activity of these antibiotic-loaded phage syringes against penicillin-resistant E. coli. The results demonstrate that phage syringes effectively reduce the host population compared to negative controls, including free penicillin and water. This study highlights the potential of using phage structures as antibiotic delivery vehicles, offering a novel strategy to overcome both the limitations of small-molecule antibiotics and phage therapy.