Loading of 3WJ/LUC2-siRNA and 3WJ/siSur into FA/EV and EGFR/EV

Exosomes (EVs) (100 μg of total protein) and RNA nanoparticles harboring LUC2 siRNA or Survivin siRNA (10 μg) were mixed in 100 μL of PBS with 10 μL of Exo-Fect Exosome transfection solution (System Biosciences), followed by a heat-shock protocol to complete the RNA nanoparticle loading. Cholesterol-modified 3WJ/FA or cholesterol-modified 3WJ/EGFR (or 3WJ) nanoparticles were incubated with RNA-loaded exosomes for decoration at an average ratio of 5,000:1 (RNA:exosome) at 37°C for 45 min and then left on ice for 1 h to prepare the FA/EV and EGFR/EV. To purify RNA-decorated EVs, 400 μL of RNA-decorated EVs was washed with 5 mL of PBS in a SW-55 tube that contained 20 μL of 60% iodixanol cushion and spun at 100,000 × g for 70 min at 4°C. All the pellets in the cushion were collected and suspended in 400 μL of sterile PBS for further use.