Apramycin

The minicells were extracted and purified by gradient centrifugation in 20% sucrose solution. The ΔEcN cells harboring the pET28a-pHLIP or pET28a-Lux plasmids were cultured in 500 mL of LB medium at 37 ℃ overnight. The bacteria and minicells were harvested via centrifugation at 10000 ×g for 20 min. The pellets were resuspended in buffered saline gelatin (BSG) and vortexed for 10 min at maximum speed. The suspensions were first centrifuged for 10 min at 2000 ×g to remove most bacteria, and the supernatants were collected after centrifugation at 10000 ×g for 30 min. The obtained pellets were resuspended and cultured in LB solution containing 25 mg/L nalidixic acid and 50 μg/mL apramycin (Sangon Biotech, Shanghai, China) at 37 °C for 1 h to kill the residual bacteria. The resultant solutions were centrifuged at 10000 ×g for 30 min and washed using BSG. The obtained minicells were spread on an LB plate and cultured overnight to detect possible contamination. Finally, the minicell pellet was lyophilized in trehalose for long-term storage and reconstituted in PBS for in vitro and in vivo experiments.