Nano f plus

Dox (Tocris Bioscience, Bristol, UK) was diluted in ultrapure water to make a stock of 10 mM. Subsequent dilutions of the 10 mM stock were carried out using PBS. Purified exosomes were incubated with Dox at various concentration ratios for 4 h at 25°C. In one set of experiments, Dox was loaded on exosomes by electroporation in three conditions: 500 V, 10 pulses and 20 ms; 750 V, 10 pulses and 20 ms; 1000 V, 2 pulses and 10 ms, using the Neon™ transfection system (Thermo Fisher Scientific). The Exo-Dox mixture was washed twice with PBS using 30 kDa MWCO Pierce protein concentrators (Thermo Fisher Scientific) to remove excess Dox. Loaded Dox was quantified against a standard curve starting at 20 ng/μl using the Nano F Plus (Tecan, Switzerland; excitation = 485 nm ± 20 nm; emission = 535 nm ± 25 nm). The loading efficiency was calculated as the average percentage of the loaded drug (ng) over the total drug input (ng). Freshly loaded exosomes were used immediately for exosome uptake and MTT cell viability assays.

On a 96-well plate, MCF-7 cells were seeded at 7000 cells/well and MDA-231 cells were seeded at 15,000 cells/well. Cells were treated with 8 points of twofold diluted free Dox or Exo-Dox, with the highest concentrations being 1.2 ng/μl and 6 ng/μl for MCF-7 and MDA-231 cells, respectively. A CyQUANT MTT cell proliferation assay kit (Invitrogen, CA, USA) was used according to the manufacturer's instructions. Briefly, 10 μl of 12 mM MTT was added to wells containing fresh medium and incubated for 3 h at 5% CO 2 and 37°C. All but 25 ul of medium was carefully removed and 100 μl DMSO was added and incubated for 10 min at 37°C to dissolve the insoluble formazan. All wells were gently resuspended to mix and the absorbance was read at 492 nm using the Nano F Plus (Tecan, Zürich, Switzerland).